ES2333880T3 - POLYPEPTIDES AND NUCLEIC ACIDS CODING THEM. - Google Patents

Abstract

Isolated nucleic acid encoding a PRO245 polypeptide, or complement thereof, in which the polypeptide: (i) comprises amino acids 1 to 312 of Figure 2 (SEQ ID No. 64) or a mature form thereof; (ii) comprises a truncated or naturally secreted form, a natural variant form or a natural allelic variant of the polypeptide of Figure 2 (SEQ ID No. 64) and inhibits the proliferation of endothelial cell growth stimulated by VEGF, with the condition that the polypeptide does not consist of amino acids 1-288 or 21-288 of Figure 2 (SEQ ID No. 64); (iii) has at least 80% identity in the amino acid sequence with the amino acid sequence of Figure 2 (SEQ ID No. 64) and inhibits the proliferation of endothelial cell growth stimulated by VEGF, wherein said sequence identity is determined over the entire length of said sequence, with the proviso that the polypeptide does not consist of amino acids 1-288 or 21-288 of Figure 2 (SEQ ID No. 64); or (iv) has at least 95% identity in the amino acid sequence with the amino acid sequence of Figure 2 (SEQ ID No. 64) and inhibits the proliferation of endothelial cell growth stimulated by VEGF, in which said sequence identity is determined over the entire length of said sequence.

Description

Polypeptides and nucleic acids that encode

Field of the Invention

The present invention relates generally to the identification and isolation of novel DNA and production recombinant of novel polypeptides encoded by that DNA.

Background of the invention

Extracellular and transmembrane proteins they play important roles in information, differentiation and maintenance of multicellular organisms. The fate of many individual cells, for example, proliferation, migration, differentiation or interaction with other cells, normally it is directed by the information received from other cells and / or in the immediate environment. This information is often transmitted. secretion polypeptides (eg, mitogenic factors, survival factors, cytotoxic factors, differentiation, neuropeptides or hormones) which are, in turn, received and interpreted by various cell receptors or transmembrane proteins These secretion polypeptides or molecules signaling pass through the cell secretion pathway to reach its place of action in the extracellular environment, normally in a transmembrane receptor protein.

Secretion proteins have several industrial applications, including their use as products Pharmaceutical, diagnostic, biosensors and bioreactors. In fact, the majority of protein drugs available here, such as thrombolytic agents, interferons, interleukins, erythropoietins, Stimulation factors of colonies and several other cytokinins, They are secretion proteins. Its receptors, which are proteins transmembrane, they also have a potential as therapeutic agents or diagnostic Immunoadhesin receptors, for example, are can be used as therapeutic agents to block the receptor / ligand interaction. Transmembrane proteins can be also employ for the selection of potential or small peptides receptor / ligand interaction inhibitor molecules relevant. Such transmembrane proteins and cell receptors, include, but are not limited to, cytokinins, receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions and adhesion molecules cell as selectins and integrins. Signal transduction that regulate cell growth and differentiation is regulated partly by the phosphorylation of several cellular proteins. The protein tyrosine kinases, enzymes that catalyze that process, They can also act as receptors for growth factors. Some examples include the growth factor receptor of fibroblasts and the nerve growth factor receptor.

Efforts are being made both industrially and academically to identify new, native transmembrane receptor secretion proteins. Many of the efforts are focusing on the selection of recombinant mammalian DNA libraries to identify novel coding and transmembrane protein coding sequences. Some examples of selection procedures and techniques are described in the literature [see for example, Klein et al., Proc. Nati Acad. Sci ., 93 : 7108-7113 (1996)]; U.S. Patent No. 5,536,637].

In the present specification we We describe the identification and characterization of polypeptides of novel secretion and transmembrane and novel nucleic acids that encode those polypeptides.

PRO245

Some of the most important proteins involved in the regulation and modulation of cellular processes described above are the enzymes that regulate the levels of protein phosphorylation in the cell. For example, it is known that the signal transduction that regulate growth and cell differentiation is regulated, at least in part, by phosphorylation and dephosphorylation of several cellular proteins. The enzymes that catalyze these processes include proteins kinases, which act by phosphorylating various cellular proteins, and the phosphatases proteins, which act by eliminating phosphate residues of several cellular proteins. In this way, the balance of Phosphorylation level of proteins in the cell is mediated by the relative activities of these two types of enzymes.

Although many enzymes have been identified protein kinase, the physiological role played by many of these Catalytic proteins must still be elucidated. However it is fine known, that a set of known protein kinases they act by phosphorylating tyrosine residues in proteins, leading in this way to a variety of different effects. Maybe most importantly, there has been great interest in proteins tyrosine kinase, since the discovery that many products oncogenes and growth factors possess intrinsic activity of protein tyrosine kinase. Therefore, there is a need for identify new members of the tyrosine protein family kinase

Given the physiological importance of proteins kinases, efforts are being made by the industry and Scientists to identify new native protein kinases. Many of these efforts focus on library screening of recombinant mammalian DNA to identify the sequences encoders for new protein kinases. At the moment invention describes the identification and characterization of new polypeptides that exhibit tyrosine protein homology kinase, referred to herein as polypeptides PRO245.

Summary Description of the Invention PRO245

Applicants have identified a clone of CDNA encoding a new polypeptide, in which the polypeptide is denominated in the present application as "PRO245".

In one embodiment, the present invention provides an isolated nucleic acid molecule as it is defined in the claims comprising a DNA encoding a PRO245 polypeptide. In one aspect, the isolated nucleic acid comprises the DNA encoding the PRO245 polypeptide that has the amino acid residues 1 to 312 of Figure 2 (SEQ ID No. 64), or is complementary to said coding nucleic acid sequence, and remains stably bound to it under conditions so less than moderate astringency, and optionally, high astringency.

In another embodiment, the present invention provides an isolated PRO245 polypeptide as defined in the claims. In particular, the present invention provides an isolated native sequence PRO245 polypeptide, which in a embodiment, includes an amino acid sequence comprising the residues 1 to 312 of Figure 2 (SEQ ID. No. 64).

Additional realizations

In other embodiments of the present invention, the invention provides vectors as defined in the claims. A host cell comprising any of said vectors is also provided. By way of example, the host cells can be CHO, E. coli or yeast cells. A method is also provided for producing any of the polypeptides described above or below, as defined in the claims and comprises host cell culture under conditions suitable for expression of the desired polypeptide and recovery of the desired polypeptide from cell culture. .

In other embodiments, the present invention provides chimeric molecules as defined in the claims. An example of said chimeric molecule comprises any of the polypeptides described above or below fused to a tag epitope sequence or an Fc region of an immunoglobulin

In another embodiment, the present invention provides an antibody as defined in the claims, which specifically binds to any of the polypeptides described above or below. Optionally, the antibody in a monoclonal antibody.

Brief description of the drawings

Figure 1 shows a nucleotide sequence (SEQ ID No. 63) of a native sequence PRO245 cDNA, in which SEQ ID. No. 63 is a clone referred to in the present invention as "UNQ219" and / or "DNA35638-1141".

Figure 2 shows the amino acid sequence (SEQ ID No. 64) derived from the coding sequence of SEQ ID. No. 63 shown in Figures 1.

Detailed description of the preferred embodiments I. Definitions

The terms "PRO polypeptide" and "PRO" are used herein and when they are immediately followed by a numerical denomination refers to several polypeptides, in which the full denomination (ie, PRO / number) refers to specific polypeptide sequences such as It is described herein. The terms "PRO polypeptide / number" and "PRO / number" as used in the Present specification encompasses sequence polypeptides native and polypeptide variants (defined in more detail in the present specification). PRO polypeptides described in the present specification can be isolated from several sources, such as from human tissue types or from another source, or prepared with recombinant synthetic procedures.

A "native sequence PRO polypeptide" comprises a polypeptide that has the same amino acid sequence  than the corresponding natural PRO polypeptide. Such polypeptides Native sequence PRO can be isolated from nature or they can be produced by recombinant synthetic means. The term "native sequence PRO polypeptide" specifically encompasses truncated natural or secretion forms of the PRO polypeptide specific (for example, alternative forms of help) and variants natural allelics of the polypeptide. In an embodiment of the invention, the native sequence PRO245 polypeptide is a PRO245 polypeptide of mature or length native sequence complete comprising amino acids 1 to 312 of Figure 2 (SEC ID No. 64).

"PRO polypeptide variant" means an active PRO polypeptide as defined above or at continuation that has at least about 80% identity amino acid sequence with the PRO polypeptide sequence of full length native sequence as described in the Present descriptive report. Such variants of the PRO polypeptide include, for example, PRO polypeptides in which they are added, or remove one or more amino acid residues, at the amino (N) or carboxyl (C) terminal of the native amino acid sequence of full length Usually, a PRO polypeptide variant will have at least about 80% percent identity of amino acid sequence, more preferably at least approximately 90% amino acid sequence identity e even more preferably at least about 95% of amino acid sequence identity with the sequence of amino acids of the native length amino acid sequence complete as described herein.

The "percentage (%) of sequence identity of amino acids "with respect to PRO polypeptide sequences identified in this specification is defined as the percentage of amino acid residues in a candidate sequence that they are identical to the amino acid residues of the sequence specific PRO polypeptide, after aligning the e sequences introduce disparity spaces, if necessary, to achieve the maximum percentage of sequence identity, and without considering no conservative substitution as part of the identity of sequence. The alignment for purposes of determining the percent amino acid sequence identity can be achieved in various ways accessible to those skilled in the art, by example, using publicly available software as the BLAST, ALIGN or Megalign (DNASTAR) programs. The program of Preferred alignment is BLAST. Those skilled in the art can determine the appropriate parameters for the measurement of alignment, including any algorithm needed to achieve maximum alignment over the entire length of the sequences They are comparing.

The "percentage (%) of sequence identity of nucleotides "with respect to the nucleotide sequences that encode the PROs identified in this specification defined as the percentage of nucleotides in a sequence candidate that are identical to the nucleotides of the sequence of PRO amino acids of interest, after aligning the e sequences introduce disparity spaces, if necessary, to achieve the maximum percentage of sequence identity, and without considering no conservative substitution as part of the identity of sequence. The alignment for purposes of determining the percent nucleotide sequence identity can be achieved in various ways accessible to experts in the field, by example, using publicly available software such as the BLAST, ALIGN or Megalign (DNASTAR) programs. The program of preferred alignment is BLAST. Subject matter experts they can determine the appropriate parameters for the measurement of alignment, including any algorithm needed to achieve maximum alignment over the entire length of the sequences They are comparing.

"Isolated", when used to describe the various polypeptides described herein, means that it has been identified and separated and / or recovered from a component of its natural environment. The contaminating components of their natural environment are materials that can typically interfere with the diagnosis or therapeutic uses of the polypeptide, and which may include enzymes, hormones, and other protein or non-protein solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer (rotary cup), or (2) to homogenize with SDS-PAGE (polyacrylamide gel with SDS) under non-reducing or reducing conditions using Coomassie Blue or, preferably, silver staining. The isolated polypeptide includes polypeptide in situ in the recombinant cells, since at least one component of the natural environment of the PRO polypeptide will not be present. Usually, however, the isolated polypeptide will be prepared at least with a purification step.

A PRO polypeptide nucleic acid "isolated" is a nucleic acid molecule that is identified and  separates at least one contaminant nucleotide molecule with the which is normally associated in the natural source of the acid PRO polypeptide nucleic. A nucleic acid molecule PRO isolated polypeptide is different from the form or environment in the It is found in nature. Nucleic acid molecules PRO isolated polypeptide differ, therefore, from the specific PRO polypeptide nucleic acid molecule that exists in natural cells. However, an acid molecule PRO isolated polypeptide nucleic acid includes acid molecules PRO polypeptide nucleic contained in cells that normally express the PRO polypeptide where, for example, the molecule of nucleic acid is in a different chromosomal location than the of natural cells.

"Southern analysis" or "Southern blotting" is a procedure whereby the presence of DNA sequences in a digestion with DNA restriction endonucleases or a composition containing DNA is confirmed by hybridization with an oligonucleotide or a DNA fragment known tagging. Southern analyzes typically involve an electrophoretic separation of DNA digestions in agarose gels, denaturation of DNA after electrophoretic separation, and the transfer of DNA to a support membrane of nitrocellulose, nylon or other suitable material for analysis with a probe labeled with an enzyme, biotinized or radioactively labeled as described in sections 9.37-9.52 of Sambrook et al., " Molecular cloning: A Laboratory Manual " (New York: Cold Spring Harbor Laboratory Press, 1989).

"Northern analysis" or "Northern blotting" is a procedure used to identify RNA sequences that hybridize with a probe known as an oligonucleotide, a DNA fragment, cDNA or its fragments, an RNA fragment. The probe is labeled with a radioactive isotope as P32, or by biotinization, or with an enzyme. The RNA to be analyzed is normally electrophoretically separated on an agarose or polyacrylamide gel, transferred to a nitrocellulose, nylon or other suitable material membrane and hybridized with a probe, using well-known usual techniques such as those described. in sections 7.39-7.52 of Sambrook et al., supra .

The term "control sequences" refers to to DNA sequences necessary for the expression of a sequence encoder operatively linked in a host organism particular. The appropriate control sequences for prokaryotes, by example, include a promoter, optionally a sequence operator, and a ribosome binding site. It is known that the cells Eukaryotes use promoters, polyadenylation signals and enhancers

A nucleic acid is "bound Operationally "when placed in a functional relationship with Another nucleotide sequence. For example, DNA for a presequence or a secretion guide sequence is operably linked  to the DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a sequence coding if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a sequence coding if it is placed so that it facilitates translation. Generally, "operably linked" means that DNA sequences that are joining are contiguous, and in the case of the secretory, contiguous and guideline sequences of reading. However, enhancers do not have to be contiguous. Binding is done by ligation at restriction sites convenient. If such sites do not exist, adapters are used synthetic oligonucleotides or binding DNA according to practice conventional.

The term "antibody" is used in the broader sense and specifically encompasses monoclonal antibodies  unique anti-polypeptide PRO (including antibodies agonists, antagonists and neutralizers) and compositions of PRO anti-polypeptide antibodies with specificity polyepitopic The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of antibodies substantially homogeneous, that is, the individual antibodies comprising the population are identical except for possible natural mutations that They may be in minor quantities.

"Active" or "activity" for purposes of this specification refer to forms of the PRO polypeptide that maintain the biological activities and / or Immunological specific native or natural polypeptide.

"Treatment" or "treatment" is refers to both medical treatment and preventive measures or prophylactic Those who need treatment include those who they already have the disorder, like those prone to it, in which We must prevent the disorder.

"Mammal" for the purposes of treatment refers to any animal classified as a mammal,  including humans, pets and farm animals, and zoo, sports or companion animals, such as sheep, dogs, Horses, cats, cows and the like. Preferably, the mammal of The present specification in a human.

"Vehicles" as used herein Descriptive report include pharmaceutically acceptable vehicles,  excipients, or stabilizers that are not toxic to the cell or mammal exposed to the doses and concentrations used. Often the physiologically acceptable vehicle is an aqueous solution buffered Some examples of physiological vehicles Acceptable include buffers such as phosphate, citrate, and other acids organic; antioxidants including ascorbic acid; polypeptides low molecular weight (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids like glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrans; chelating agents such as EDTA; polyols such as mannitol or sorbitol; salt forming counterions such as sodium; I nonionic surfactants such as TWEEN?, polyethylene glycol (PEG) and PLURONICS ™.

The term "agonist" is used to make reference to peptide and non-peptide analogs of the polypeptides  Native PRO (where native PRO polypeptide refers to PRO propolypeptide, PRO prepolypeptide, PRO prepropolypeptide or mature PRO polypeptide) of the present invention and to antibodies that specifically bind to such native PRO polypeptides, with the condition that they maintain at least one biological activity of the mature PRO polypeptide. Preferably, the agonists of the The present invention maintains the recognition properties of Qualitative binding and receptor activation properties of the mature PRO polypeptide.

The term "antagonist" is used to make reference to a molecule that inhibits a biological activity of the mature PRO polypeptide of the present invention in which Mature PRO polypeptide refers to PRO propolypeptide, PRO prepolypeptide, PRO prepropolypeptide or mature PRO polypeptide. Preferably, the antagonists herein descriptively inhibit the binding of a mature PRO polypeptide of the present invention An "antagonist" PRO polypeptide is a molecule that prevents or interferes with an antagonistic effector function  PRO (for example, a molecule that prevents or interferes with binding and / or activation of a PRO polypeptide receptor by a PRO polypeptide). Such molecules can be selected by their ability to completely inhibit receptor activation of the PRO polypeptide controlling the binding of a PRO polypeptide in presence and absence of the test antagonist molecule, by example. An antagonist of the invention also encompasses a antisense polynucleotide against the PRO polypeptide gene, such antisense polynucleotide blocks transcription or translation of the PRO polypeptide gene, thereby inhibiting its expression and biological activity

"Restrictive conditions" means (1) the use of solutions of low ionic concentration and high wash temperature, for example, 0.015 M sodium chloride; 0.0015 M sodium citrate; 0.1% sodium dodecyl sulfate at 50 ° C, or (2) employment during hybridization of an agent denaturing, such as formamide, for example, 50% formamide (v / v) with 0.1% bovine serum albumin; 0.1% Ficol; 1 of polyvinylpyrrolidone; 50 nM sodium pyrophosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C. Other example is to use 50% formamide, 5X SSC (0.75 M NaCl, citrate 0.075 M sodium), 50 mM sodium phosphate (pH6 / 8), 0.1% pyrophosphate sodium, Denhardt 5X solution, salmon sperm DNA sonicated (50 µg / ml), 0.1% SDS and 10% sulfate dextran at 42 ° C, with washes at 42 ° C in 0.2X SSC and 0.1% SDS. Still another example is hybridization using a buffer with 10% of dextran sulfate, SSC 2X (sodium chloride / sodium citrate) and 50% formamide at 55 ° C, followed by a highly restrictive wash consisting of 0.1X SSC containing EDTA at 55 ° C.

"Moderately restrictive conditions" are described in Sambrook et al., Supra and include the use of a wash solution and hybridization conditions (eg temperature, ionic concentration and% SDS) less restrictive than those described above. An example of moderately restrictive conditions is a condition such as overnight incubation at 37 ° C in a solution comprising: 20% formamide, 5X SSC (0.75 M NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), Denhardt 5X solution, 10% dextran sulfate, and 20 mg / ml of fragmented and denatured salmon sperm DNA, followed by washing the filter in 1X SSC at 37-50 ° C. Those skilled in the art will know how to adjust the temperature, ionic concentration, etc., as necessary to accommodate factors such as probe length and the like.

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II Compositions and Procedures of the Invention PRO245 full length polypeptides

The present invention provides sequences of newly identified and isolated nucleotides encoding polypeptides referred to in the present application are referred to as PRO245. In particular, Applicants have identified and isolated CDNA encoding a PRO245 polypeptide, as described with more detail in the following examples. Using the programs BLAST and FastA sequence alignment software, the Applicants discovered that a part of the sequence of PRO245 polypeptide amino acids have a 60% identity of amino acids with the human c-myb protein. By consequently, it is currently believed that the PRO245 polypeptide described in this application can be an identified member recently from the family of transmembrane tyrosine proteins kinase

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PRO polypeptide variants

In addition to the PRO sequence polypeptides full length native described herein descriptive, it is contemplated that variants of the PRO polypeptide. PRO polypeptide variants can be prepared introducing the appropriate nucleotide changes in the DNA of the PRO polypeptide, or synthesizing the desired PRO polypeptide. The Those skilled in the art will appreciate that amino acid changes they can alter the post-translational processes of the polypeptides PRO, such as changes in the number or position of glycosylation sites or Alterations the characteristics of anchoring to the membrane.

Variations of the full-length native sequence PRO polypeptides or in several domains of the PRO polypeptides, described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for example, in US Pat. No. 5,364,934. Variations may be substitution, deletion or insertion of one or more codons encoding the PRO polypeptide that results in the change of the amino acid sequence of the PRO polypeptide compared to the native sequence PRO polypeptide. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more domains of the PRO polypeptide. The orientation to determine which amino acid can be introduced, substituted or deleted without adversely affecting the desired activity can be found by comparing the sequence of the PRO polypeptide with that of homologous known protein molecules and minimizing the number of changes in the amino acid sequence made in regions of high homology Amino acid substitutions may result from replacing one amino acid with another that has similar structural and chemical characteristics, such as replacing a leucine with a serine, that is, conservative amino acid replacements. Insertions and deletions may optionally be in the range of 1 to 5 amino acids. The permitted variation can be determined by making systematic insertions, deletions or substitutions of amino acids in the sequence and testing the activity of the resulting variants in the in vitro assay described in the following Examples.

Variations can be made using methods known in the art such as oligonucleotide-mediated (directed) mutagenesis, systematic alanine substitution and PCR mutagenesis. Targeted mutagenesis [Carter et al., Nucl. Acids Res ., 13 : 4331 (1986); Zoller et al., Nucl. Acids Res ., 10 : 6487 (1987)], mutagenesis with caete [Wells et al., Gene , 34 : 315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans R. Soc. London SerA , 317 : 415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PRO polypeptide variant DNA.

Systematic substitution amino acid analysis can also be used to identify one or more amino acids along a contiguous sequence. Among the preferred systematic replacement amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is typically a preferred systematic substitution amino acid among this group since it eliminates the side chain beyond the beta carbon and is less likely to alter the conformation of the main chain of the variant. Alanine is also typically preferred because it is the most common amino acid. In addition, it is frequently found in both protected and exposed positions [Creighton, The Proteins , (WH Freeman and Co., NY); Chothia, J. Mol. Biol ., 150 : 1 (1976)]. If the alanine substitution does not produce adequate amounts of variant, an isosteric amino acid can be used.

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PRO Polypeptide Modifications

Covalent modifications of PRO polypeptides are included within the scope of this invention. One type of covalent modification includes reacting the target amino acid residues of the PRO polypeptide with an organic derivatizing agent that is capable of reacting with the selected side chains or the N-terminal or C-terminal residues of the PRO polypeptide. Derivatization with bifunctional agents is useful, for example to cross-link ( crosslinking ) a PRO polypeptide to a water insoluble matrix or support surface for use in the process for purifying anti-PRO polypeptide antibodies, and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane; glutaraldehyde; N-hydroxysuccinimide esters, for example, esters with 4-acidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis (succinidimylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents as methyl-3 - [(p-acidophenyl) dithio] proprioimidate.

Other modifications include the deamination of glutaminyl and asparraginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of the side chains of lysine, arginine and histidine [TE Creighton, " Proteins: Structure and Molecular Properties " Freeman and Co., San Francisco, pp. 79-86 (1983)].

Another type of covalent modification of PRO polypeptides included in the scope of this invention comprises the alteration of the native glycosylation pattern of the polypeptide. "The alteration of the glycosylation pattern native "is designed for the purposes of this report descriptive to mean eliminating one or more glycidic residues found in the native sequence of the PRO polypeptide, and / or add one or more glycosylation sites that are not present in the native sequence PRO polypeptide.

The addition of glycosylation sites to PRO polypeptide can be achieved by altering the sequence of amino acids. The alteration can be made, for example, by adding or replacing one or more serine or threonine residues at native sequence PRO polypeptide (for binding sites O-glycosidic). The amino acid sequence of PRO polypeptide can be optionally altered through changes to DNA level, particularly by mutating the DNA encoding the PRO polypeptide in preselected bases since those codons generated will be translated into the desired amino acids.

Other means of increasing the number of glycosidic residues in the PRO polypeptide is by chemical or enzymatic binding of glycosides to the polypeptide. Such procedures are described in the art, for example, in WO87 / 05330 published on September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem ., Pp. 259-306 (1981).

The elimination of glycosidic residues present in the PRO polypeptide can be achieved chemically or enzymatically or by mutational substitution of codons encoding amino acid residues that serve as a glycosylation target. Chemical deglycosylation techniques are known in the art and are described, for example, in Hakimuddin et al., Arch. Biochem. Biophys ., 259 : 52 (1987) and in Edge et al., Anal. Biochem ., 118 : 131 (1981). Enzymatic cleavage of glycidic polypeptide residues can be achieved using a variety of endoglycosidases and exoglycosidases as described in Thotakura et al., Meth. Enzymol ., 138 : 350 (1987).

Another type of covalent modification of PRO polypeptides of the invention comprise binding of the polypeptide PRO to a variety of non-protein polymers, for example, polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in the manner set forth in US Pat. No. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

The PRO polypeptides of the present invention are they can also modify so that they form a chimeric molecule comprising a PRO polypeptide fused to another sequence heterologous polypeptide or amino acid. In one embodiment, such chimeric molecule comprises a fusion of the PRO polypeptide with a tag polypeptide that provides an epitope to which a anti-label antibody can be selectively bound. The label epitope is usually placed at the amino terminal ends or PRO carboxyl terminal of the PRO polypeptide. The presence of such epitope-tagged forms allow the PRO polypeptide to be easily purified by affinity purification using a anti-label antibody or other type of affinity matrix that binds to epitope tag. In an alternative embodiment, the molecule chimeric may comprise a fusion of the PRO polypeptide with a immunoglobin or a particular region of an immunoglobin. For one bivalent form of the chimeric molecule, such fusion can be with the Fc region of an IgG molecule.

Several tag polypeptides and their respective antibodies are well known in the art. Some examples include polyhistinide (poly-His) or polyhistidinglycine (Poly-His-Gly); the HA tag polypeptide of influenza and its 12CA5 antibody [Field et al., Mol. Cell Biol ., 8: 2159-2165 (1988)]; the Myc-c label and its antibodies 8F9, 3C7, 6E10, G4, B7 and 9E10 [Evan et al., Moecular and Cellular Biology , 5 : 3610-3616 (1985)] .; and the Herpes Simplex virus (gD) glycoprotein D tag and its antibody [Paborsky et al., Protein Engineering , 3 (6) : 547-553 (1990)] Other tag polypeptides include the Flag peptide [Hopp et al., Biotechnology , 6 : 1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science , 225 : 192-194 (1992)]; an α-tubulin epitope peptide [Skinner et al., J. Biol. Chem ., 266 : 15163-15166 (1991)] and the peptide tag of the T7 1st gene protein [Lutz-Freyermuth et al., Proc. Nati Acad. Sci. USA 87 : 6393-6397 (1990)].

In WO99 / 14328, from which the present is derived request, a more detailed description of the modification of another PRO317 polypeptide.

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PRO Polypeptide Preparation

The following description mainly describes the production of PRO polypeptides by culturing cells transformed or transfected with a vector containing the nucleic acid of the desired PRO polypeptide. It is contemplated, of course, that alternative methods, well known in the art, can be used to prepare the PRO polypeptide. For example, the sequence of the PRO polypeptide, or portions thereof, can be produced by direct peptide synthesis using solid phase techniques [see for example, Stewart et al., Solid-Phase Peptide Synthesis , WH Freeman and Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc ., 85 : 2149-2154 (1963)]. In vitro protein synthesis can be performed using manual or automatic techniques. Automatic synthesis can be achieved, for example, using an Applied Biosystems peptide synthesizer (Foster City, CA) using the manufacturer's instructions. Several portions of the desired PRO polypeptide can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full length PRO polypeptide.

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A. Isolation of DNA encoding PRO polypeptides

The DNA encoding the PRO polypeptides is you can get from a cDNA library prepared from a tissue that is believed to possess the desired PRO polypeptide mRNA at a detectable level Therefore, the PRO polypeptide DNA human can be conveniently obtained from a cDNA library prepared from a human tissue, as described in the Examples The gene encoding the PRO polypeptide can be obtained also from genomic libraries or by synthesis of oligonucleotides

The libraries can be investigated with probes (such as antibodies against the desired PRO polypeptide or oligonucleotides of at least about 20-80 base pairs) designed to identify the gene of interest or the protein it encodes. Research of the cDNA or genomic library with the selected probe can be carried out using standard procedures, as described in Sambrook et al., " Molecular Cloning: A Laboratory Manual " (New York : Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding the desired PRO polypeptide is to use the PCR methodology [Sambrook et al., Supra ; Dieffenbach et al., " PCR Primer: A Laboratory Manual " ( PCR Primer: Laboratory Manual ; New York: Cold Spring Harbor Laboratory Press, 1995)].

The following Examples describe techniques to investigate a cDNA library. The oligonucleotide sequences selected as probes must be of sufficient length and accurate enough to minimize false positives. The oligonucleotide is preferably labeled so that it can be detected once hybridized to the DNA in the library being investigated. Labeling procedures are well known in the art, and include the use of radioactive labels such as ATP labeled with P 32, biotinization or enzymatic labeling. Hybridization conditions, including moderately restrictive and very restrictive conditions, are provided in Sambrook et al., Supra .

The sequences identified in such library research procedures can be compared and align with other known sequences deposited and available in public databases such as GenBank or other databases of private sequences Sequence identification (level amino acid or nucleotide) within the defined regions of the molecule or for the entire length of the sequence can be determined through sequence alignment using a program software such as BLAST, ALIGN, DNAstar and INHERIT that employs several algorithms to measure homology.

Nucleic acid having the sequence encoding the protein can be obtained by investigating the cDNA or genomic library using the deduced amino acid sequence described herein for the first time and, if necessary, using conventional primer extension procedures that are described in Sambrook et al., supra , to detect precursors and mRNA processing intermediaries that may not have been retrotranscribed to cDNA.

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B. Selection and transformation of host cells

Host cells are transfected or transformed with the expression or cloning vectors described herein for the production of PRO polypeptide and cultured in conventional modified nutritional media as appropriate to induce promoters, select transformants or amplify genes. that encode the desired sequences. Those skilled in the art can select the culture conditions, such as media, temperature, pH and the like, without excessive experience. In general, in " Mammalian Cell Biotechnology a Practical Approach ", M. Butler, ed. (IRL Press, 1991) and Sambrook et al., Supra , you can find the principles, protocols and practical techniques to maximize the productivity of cell cultures.

Those of ordinary skill in the art know very well the methods of transfection, for example, CaPO4 and electroporation. Depending on the host cell that is used, the transformation is performed using usual techniques appropriate for such cells. Calcium treatment, which employs calcium chloride, as described in Sambrook et al., Supra , or electroporation is generally used for prokaryotes or other cells that contain considerable barrier cell walls. Infection with Agrobacterium tumefaciens is used for the transformation of certain plant cells, as written in Shaw et al., Gene , 23 : 315 (1983) and in WO89 / 05859 published June 29, 1989. For cells of mammalian without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb., Virology , 53 : 456-457 (1978) can be employed. The general aspects of the transformations of mammalian host cell systems have been described in US Pat. No. 4,339,216. Yeast transformations are typically carried out according to the procedure of Van Solingen et al., J. Bact ., 130 : 946 (1977) and Hsiao et al., Proc. Nati Acad. Sci. (USA) , 76 : 3829 (1979). However, other methods can also be used to introduce DNA into cells, such as by nuclear microinjection, electroporation, fusion of bacterial protoplasts with intact cells, or polycations, for example, polybrene, polynithin. For various mammalian cell transformation techniques, see Keown et al., Methods in Enzimology , 185 : 527-537 (1990) and Mansour et al., Nature , 336 : 348-352 (1988).

Suitable host cells for cloning or expressing vector DNA herein include prokaryotes, yeasts or higher eukaryotic cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram negative or Gram positive organisms, for example Enterobacteria such as E. coli . Several strains of E. coli are publicly available, such as E. coli strain K12 MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,357); E. coli strain W3110 (ATCC 53,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include enterobacteria such as Escherichia , for example, E. coli, Enterobacter, Erwinia, Kleibsella, Proteus, Salmonella , for example, Salmonella typhymurium, Serratia , for example, Serratia marcescens, and Sighella , as well as Bacilli such as, B subtillis and B. licheniformis (for example, B. licheniformis 41P described in DD 266,710 published April 12, 1989), Pseudomonas as P. aureginosa , and Streptomyces . Several strains of E. coli are publicly available, such as E. coli strain K12 MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,357); E. coli strain W3110 (ATCC 53,325) and K5 772 (ATCC 53,635). These examples are illustrative rather than limiting. Strain W3110 is a host cell or parenteral cell particularly preferred because it is a common host strain for fermentations with recombinant DNA products. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example strain W3110 can be modified to perform a genetic mutation in the genes encoding endogenous host proteins, with examples of such hosts including E. coli W3110 strain 1A2, which has the complete genotype tonA; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3, E. coli W3110 strain 27C7 (ATCC 55.244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT karl; E. coli W3110 strain 37D6 (ATCC 55.244), which has the complete genotype tonA ptr3phoA E15 (argF-lac) 169 degP ompT rbs7 ilvG kan r; E. coli W3110 strain 40B4 which is strain 37D6 with a degP deletion mutation without kanamycin resistance; and an E. coli strain that has a mutant periplasmic protease described in US Pat. No. 4,946,783 published August 7, 1990. Alternatively, other in vitro cloning procedures are suitable, for example PCR or other reactions with nucleic acid polymerase.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable as cloning or expression vector hosts encoding the PRO polypeptide. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature , 290 : 140 [1981]; (EP139,383 published May 2, 1985); Kluiveromyces Hosts (US Patent No. 4,943,529; Fleer et al. ., Bio / Technology , 9 : 968-975 (1991)), such as K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol ., 737 [1993]), K. fragilis (ATCC 12.244), K. bulgaricus (ATCC16.045), K. wickeramii (ATCC 24.178), K. waltii (ATCC 56,500), K. drosophylarum (ATCC 36.906; Van dern Berg et al., Bio / Technollogy , 8 : 135 (1990)); K. thermotolerans and K. marxianus; yarrowia (EP402,226) Pichia pastoris (EP183,070; Sreekrishna et al., J. Basic Microbiol ., 28 : 265-278 [1988]) ; Candida; Trichoderma reesia (document EP244,234); Neurospora crassa (Case et al., Proc. Nati. Acad. Sci. USA , 76 : 5259-5263 [1979]; Schwanniomyces as Schwanniomyces occidentalis (document EP394. 538 published October 31, 1990); and filamentous fungi as, by axis mplo Neurospora, Penicillium, Tolyplocadium (WO91 / 00357 published on January 10, 1991), and aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys Res. Commun ., 112 : 284-289 [1983]); Tiburn et al., Gene , 26 : 205-221 [1983]; Yelton et al., Proc. Nati Acad. Sci USA , 81 : 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J. 4 : 475-479 [1985]. Methylotrophic yeasts are suitable herein but not limited to, yeast capable of growing in selected methanol among the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis and Rhodotorula . A list of specific species that are examples of this kind of yeast can be found in C. Anthony, "The Biochemistry of Methylotrophs "(Biochemistry of the Methylotrophs), 269 (1982).

Suitable host cells for the expression of glycosylated PRO polypeptides are derived from multicellular organisms. Some examples of invertebrate cells include insect cells such as Drosophyla S2 and Spodoptera Sf9, as well as plant cells. Some examples of useful mammalian host cells include Chinese hamster ovary (CHO) cells and COS cells. More specific examples include CV1 monkey kidney line transformed with the SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension cultures, Graham et al., J. Gen Virol ., 36 : 59 (1977)); Chinese hamster cells / -DHFR (CHO, Urlaub and Chasin, Proc. Nati. Acad. Sci. USA , 77 : 4216 (1980)); mouse Sertoli cells (YM4, Mather Biol Reprod ., 23 : 243-251 (1980)); human lung cells (W138, ATCC CCL 75); liver cells (Hep G2, HB 8065); and mouse mammary tumor cells (MMT 060562, ATCC CCL51). It is assumed that the selection of the appropriate host cell depends on the experience in the field.

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C. Selection and use of replicative vectors

The nucleic acid (for example cDNA or DNA genomic) encoding a desired PRO polypeptide can be introduce into a replicative vector for cloning (amplification of DNA) or for expression. There are several vectors publicly available. The vector can be, for example, in the form of plasmid, cosmid, viral particle or phage. The sequence of Appropriate nucleotides can be introduced into the vector by Various procedures In general, DNA is introduced into a site (s) of a restriction endonuclease appropriate using techniques known in the art. The Vector components generally include, but are not limited to, one or more signal sequences, an origin of replication, one or more marker genes, an enhancer element, a promoter and a termination sequence of transcription. The construction of suitable vectors containing one or more of these components employs usual ligation techniques that experts in the matter know.

The PRO polypeptide of interest can be recombinantly produced not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which can be a signal sequence or other polypeptide having a specific cut-off site at the N-terminal end of the mature or polypeptide protein. In general, the signal sequence may be a component of the vector, or it may be part of the DNA of the PRO polypeptide that is introduced into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillase, 1pp, or thermostable enterotoxin II guide sequences. For secretion in yeast the signal sequence, for example, the guiding sequence of the yeast invertase, the guiding sequence of the alpha factor (including the α factors of Saccharomyces and Kluiveromyces , described last in US Pat. No. 5,010,182), or the acid phosphatase guide sequence, the C. albicans glucoamylase guide sequence (EP362,179 published April 4, 1990), or the signal sequence described in published WO90 / 13646 on November 15, 1990. In the expression of mammalian cells, signal sequences can be used to direct the secretion of the protein, as signal sequences of secretion polypeptides of the same or related species, as well as viral secretion guide sequences.

Both expression vectors and those of cloning contain a nucleotide sequence that causes the vector can be replicated in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeasts and viruses. The origin of replication of plasmid pBR322 is suitable for most gram negative bacteria, the origin of the 2 µm plasmid is suitable for yeasts and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.

Expression and cloning vectors will contain typically a selection gene, also called a marker of selection. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, by example, ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic differences, or (c) provide Critical nutrients not available in complex media, for example the gene encoding the D-alanine racemase for bacilli.

An example of suitable selection markers for mammalian cells are those that enable the identification of competent cells to carry the PRO polypeptide nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when a wild DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described in Urlaub et al., Proc. Nati Acad. Sci. USA , 77 : 4216 (1980). A suitable selection gene for use in yeasts is the trp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature , 282 : 39 (1979); Kingsman et al., Gene , 7 : 141 (1979; Tschemper et al., Genes , 10 : 157 (1980)]. The trp1 gene provides a selection marker for a mutant strain of yeast that lacks the ability to grow in Tryptophan, for example ATCC No. 44076 or PEP4-1 [Jones, Genetics , 85 : 12 (1977)].

Expression and cloning vectors normally contain a promoter operably linked to the nucleotide sequence of the PRO polypeptide to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems [Chang et al., Nature , 275 : 615 (1978); Goeddel et al., Nature , 281 : 544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system, [Goedel, Nucleic Acid Res ., 8 : 4057 (1980); EP36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Nati Acad. Sci. USA , 80 : 21-25 (1983)]. Promoters for use in bacterial systems will also contain a Shine-Dalgarno (SD) sequence operatively linked to the DNA encoding the desired PRO polypeptide.

Some examples of promoter sequences suitable for use with yeast host cells include promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem ., 255 : 2073 (1980)], or other glycolytic enzymes [Hess and col., J. Adv. Enzyme Reg . 7 : 149 (1968)]; Holland, Biochemistry , 17 : 4900 (1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosaphosphate isomerase, phosphogase and phosphate isomerase, phosphate glucokinase

Other yeast promoters, which are promoters inducible that have the additional advantage of having controlled the transcription through growth conditions, are the regions promoters for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradation enzymes associated with the metabolism of nitrogen, metallothionein, glyceraldehyde-3-phosphate dehydrogenase and enzymes responsible for the use of the maltose and galactose. Vectors and promoters suitable for Use in yeast expression is described in more detail in the EP73,657.

Transcription of the PRO polypeptide from vectors in mammalian host cells is controlled by example, by promoters obtained from virus genomes such as polyoma virus or avian smallpox virus (document UK2.211.504 published July 5, 1989), adenovirus (as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus a, hepatitis B virus and Ape 40 (SV40), from heterologous mammalian promoters, by example, the actin promoter or a promoter of a immunoglobulin, and heat shock promoters, provided that promoters are compatible with cell systems Guest.

The transcription of a DNA encoding the desired PRO polypeptide in higher eukaryotes can be increase by introducing an enhancer sequence in the vector. Enhancers are elements that act in cis, usually of approximately 10 to 300 bp, which act on a promoter for Increase your transcript. Many enhancer sequences are They know very well today for mammalian genes (globin, elastase, albumin, α-fetoprotein e insulin). Typically, however, an enhancer of a eukaryotic cell virus. Some examples include the enhancer SV40 at the late end of the origin of replication (bp 100-270), the enhancer of the early promoter of cytomegalovirus, the polyoma enhancer or at the late end of the origin of replication, and adenovirus enhancers. He enhancer can be cut and moved to the 5 'or 3' position of the sequence encoding the PRO polypeptide, but is located preferably at the 5 'site of the promoter.

Expression vectors used in cells eukaryotic host (yeasts, fungi, insects, plants, animals, human or nucleated cells of another multicellular organism) they will also contain sequences necessary for the termination of the transcription and to stabilize mRNA. Such sequences are commonly available at the 5 'end and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain transcribed nucleotide segments as polyadenylated fragments in the untranslated portion of the mRNA that encodes PRO polypeptides.

Still other methods, vectors and host cells suitable for adaptation for the synthesis of PRO polypeptides in recombinant vertebrate cell cultures are described in Gething et al., Nature , 293 : 620-625 (1981); Mantei et al., Nature , 281 : 40-46 (1979); EP117.060 and EP117.058.

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D. Gene amplification / expression detection

Gene amplification and / or expression can be measured directly in a sample, for example, with a conventional Southern blot, Northern blot to quantify mRNA transcription [Thomas, Proc. Nati Acad. Sci. USA , 77 : 5201-5205 (1980)], spot transfer (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies that recognize specific double strands including double strands of DNA, double strands of RNA and double strands of hybrid DNA-RNA or double strands of DNA-protein can be used. The antibodies in turn can be labeled and the assay is carried out when the double chain is attached to the surface, so that after the formation of the double chain on the surface, the presence of antibodies bound to the chain can be detected double.

Gene expression, alternatively, can be measure by immunological procedures, such as stains tissue immunohistochemistry tissue cuts and analysis of cell cultures or blood fluids, to quantify directly the expression of the gene product. Antibodies useful for immunohistochemical staining and / or sample analysis of fluids can be monoclonal or polyclonal and can be Prepare in any mammal. Conveniently, the antibodies can be prepared against a native sequence PRO polypeptide or against a synthetic peptide based on DNA sequences provided herein or against exogenous sequences fused to the DNA of a PRO polypeptide and that encode an epitope for a specific antibody.

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E. Purification of the Polypeptide

PRO polypeptide forms can be recover from culture medium or cell lysates Guest. If they are attached to the membrane, they can be released from the membrane using a suitable detergent solution (for example Triton-X 100) or by enzymatic section. The cells used in the expression of PRO polypeptides can be broken by various physical or chemical procedures, such as cycles of freeze-thaw, sonication, break mechanical, or cell lysis agents.

It may be desired to purify PRO polypeptides from recombinant cell proteins or polypeptides. The following procedures are examples of suitable purification procedures. By fractionation in an ion exchange column; ethanol precipitation; Reverse phase HPLC; chromatography on a silica or cation exchange resin such as DEAE; chromato focus; SDS-PAGE (polyacrylamide gel with SDS); precipitation with ammonium sulfate; using gel filtration, for example, Sephadex G-75; Protein A-Sepharose columns to remove contaminants such as IgG; and columns of metal chelators to bind the tagged forms with epitopes of the PRO polypeptide. Various protein purification procedures may be known as procedures well known in the art and described for example in Deutscher, " Methods in Enzimology ", 182 (1990); Reach, " Protein Purification: Principles and Practice ", Springer-Verlag, New York (1982). The purification steps selected will depend, for example, on the nature of the production process used and the particular PRO polypeptide produced.

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Uses for PRO polypeptides

Nucleotide sequences (or their complementary) encoding the PRO polypeptides herein invention, have several applications in the biology technique molecular, including use in hybridization probes, in mapping chromosomal and gene and in the generation of antisense RNA and DNA. He nucleic acid encoding the PRO polypeptide will also be useful for the preparation of PRO polypeptides by techniques recombinants described herein.

The nucleic acid encoding the polypeptide Full-length native sequence PRO or its portions, are can be used as hybridization probes for cDNA libraries for isolate the gel from the full length PRO polypeptide or to isolate even other genes (for example, those that encode variants Natural PRO polypeptide or PRO polypeptides of others species) that have a desired sequence identity with the PRO polypeptide nucleotide sequences. Optionally, the Probe length will be approximately 20 to 50 bases. The hybridization probes can be derived from nucleotide sequences of any of the DNA molecules described herein descriptive memory or genomic sequences including promoters, enhancer elements and introns of the DNA encoding the native sequence PRO polypeptide. As an example, a selection procedure will comprise the isolation of the region PRO polypeptide gene encoder using DNA sequence known to synthesize a selected probe of approximately 40 bases Hybridization probes can be labeled with a variety of labels, including radionuclotides such as P32 or S 35, or enzymatic labels such as bound alkaline phosphatase to the probe via avidin / biotin binding systems. Can be used labeled probes that have a sequence complementary to that of the PRO specific polypeptide gene of the present invention for investigate libraries of human cDNA, genomic DNA or mRNA for identify with which members of such libraries hybridize the probe. Hybridization techniques are described in more detail in os Following Examples.

The TSEs described in this application are they can use similarly as probes, using the procedures described herein.

The probes can be used in PCR techniques to generate a sequence group to identify sequences from closely related PRO polypeptide.

Nucleotide sequences encoding a PRO polypeptide can also be used to construct hybridization probes for mapping the gene encoding the PRO polypeptide and for the genetic analysis of individuals with genetic alterations. The nucleotide sequences provided herein can be used for mapping a chromosome and specific regions of the chromosome using known techniques such as in situ hybridization, binding analysis against known chromosomal markers and hybridization library research.

PRO polypeptides can be used in analysis to identify their ligands. Similarly, you can identify inhibitors of binding interaction receptor / ligand. The proteins involved in such an interaction of binding can also be used to investigate peptides or small inhibitor or agonist molecules of the binding interaction. The research analysis can be designed to find guide compounds that mimic the biological activity of native PRO polypeptide or a ligand for the PRO polypeptide. Such research analyzes will include analyzes prepared for High performance research of chemical libraries, making them particularly suitable for identifying candidates Molecularly small pharmacological. Small molecules contemplated include synthetic organic or inorganic compounds. Analyzes can be performed in several ways, including protein-protein binding analysis, analysis of biochemical research, immunoassay and cell analysis, characterized in the art.

Nucleic acids encoding a PRO polypeptide or its modified forms can also be used to generate transgenic animals or " knock out " animals (without a certain gene) which, in turn, are useful in the development and therapeutic investigation of useful reagents. A transgenic animal (for example, a mouse or a rat) is an animal that has cells that contain a transgene, said transgene was introduced into the animal or an ancestor of the animal in a prenatal stage, for example, in an embryonic stage. A transgene is a DNA that integrates into the genome of a cell from which an animal develops. In one embodiment, the cDNA encoding a PRO polypeptide of interest can be used to clone genomic DNA encoding the PRO polypeptide according to established techniques and genomic sequences used to generate the transgenic animals that contain the cells that express the DNA encoding. the PRO polypeptide. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in US Pat. No. 4,736,866 and 4,870,009. Typically, individual cells will be targets for incorporation of a PRO polypeptide transgene with tissue specific enhancers. Transgenic animals that include a copy of a transgene encoding a PRO polypeptide introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of the DNA encoding the PRO polypeptide. Such animals can be used as test animals for reagents that are believed to confer protection against, for example, pathological conditions associated with their overexpression. According to this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological state in comparison with the untreated animals carrying the transgene would indicate a potential therapeutic intervention of the pathological state.

Alternatively, non-human homologs of the PRO polypeptides can be used to construct a knock out animal for the PRO polypeptide that has a defective or altered gene encoding the PRO polypeptides of interest as a result of homologous recombination between the endogenous gene encoding PRO polypeptides and an altered genomic DNA encoding PRO polypeptides introduced into an embryonic cell of the animal. For example, cDNA encoding a PRO polypeptide that is used to clone genomic DNA encoding the PRO polypeptide according to established techniques. A portion of the genomic DNA encoding a PRO polypeptide with another gene can be deleted or replaced as a gene encoding a selection marker that is used to control integration. Typically, several unchanged flanking DNA chilobases are included in the vector (both at the 5 'and 3' ends) [see, for example, Thomas and Capecchi, Cell , 51 : 503 (1987) for a vector description homologous recombination]. The vector is introduced into an embryonic stem cell line (for example by electroporation) and the cells in which the DNA was introduced and homologously recombined with the endogenous DNA are selected [see for example, Li et al., Cell , 69 : 915 (1992)]. The selected cells are then injected into an animal blastocyst (for example, mouse or rat) to form aggregation chimeras [see for example, Bradley, in " Teratocarcinomas and Embrionyc Stem Cells: A Practical Approach ", (Teratocarcinomas and stem cells) embryonic: A practical approach), EJ Robertson, ed. (IRL, Oxford, 1987), pp. 113-125]. A chimeric embryo can then be implanted in a suitable pseudo-pregnant female recipient animal and the embryo carried to term to create a knock out animal. The offspring carrying homologously recombined DNA in their germ cells can be identified by usual techniques and can be used to breed animals that contain homologously recombined DNA in all their cells. Knock out animals can be characterized, for example, by their ability to defend themselves against
certain pathological states and due to their development of pathological conditions due to the absence of the PRO polypeptide.

Because the PRO245 polypeptide and the nucleic acid encoding it have sequence homology with a transmembrane tyrosine kinase protein and its coding nucleic acid, probes based on the nucleotide sequence of PRO245 can be used to identify other transmembrane tyrosine kinase proteins. Soluble forms of the PRO245 polypeptide can be used as antagonists of membrane bound PRO245 activity both in vitro and in vivo . PRO245 polypeptides can be used in screening assays designed to identify agonists or antagonists of the native PRO245 polypeptide, wherein said assays can take the form of any conventional cell or biochemical type binding assay. In addition, the PRO2245 polypeptide can serve as a molecular marker for tissues in which the polypeptide is specifically expressed.

Doses and administration of PRO317, agonist of PRO317 or PRO317 antagonist in a pharmaceutical composition is described in WO99 / 14328.

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PRO Polypeptide Antibodies

The present invention further provides PRO polypeptide antibodies. Some examples of antibodies include polyclonal, monoclonal, humanized antibodies, bispecific, and heteroconjugates.

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A. Polyclonal Antibodies

PRO polypeptide antibodies can Understand polyclonal antibodies. Subject matter experts They know the procedures for preparing polyclonal antibodies. Polyclonal antibodies can be produced in a mammal, by example by one or more injections of an immunizing agent and, If desired, an adjuvant. Typically the immunizing agent and / or adjuvant will be injected into the mammal by multiple subcutaneous or intraperitoneal injections. Immunizing agent may include the PRO polypeptide or one of its fusion proteins. It may be useful to conjugate the immunizing agent to a protein that is Know that it is immunogenic in the mammal being immunized. Some examples of such immunogenic proteins include, but not they are limited to, California limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Some examples of adjuvants that can be employed include Freund's complete adjuvant and MPL-TDM adjuvant (Monophosphoryl Lipid A, synthetic trehalose dicorinomicolate). A subject matter expert without too much experience can select the immunization protocol

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B. Monoclonal antibodies

The anti-PRO polypeptide antibodies may alternatively be monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma formation procedures, such as those described in Kohler and Milstein, Nature , 256 : 495 (1975). In a hybridoma formation procedure, a mouse, hamster or other appropriate host animal is typically immunized with an immunizing agent to obtain lymphocytes that produce or are capable of producing antibodies that actively bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro .

The immunizing agent will typically include the PRO polypeptide of interest or one of its fusion proteins. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymphoid ganglion cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized line using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell [Goding, " Monoclonal Antibodies: Principles and Practice ", Academic Press, (1986 ) pp 59-103]. Immortalized cell lines are normally transformed mammalian cells, particularly myeloma cells of murine, bovine or human origin. Normally, rat or mouse myeloma cell lines are used. The hybridoma cells are cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of immortalized cells without fusing. For example, if parenteral cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas will typically include hypoxanthine, aminopterin and thymidine ("HAT medium"), whose substances prevent the growth of deficient cells in HGPRT.

Preferred immortalized cell lines are those that effectively fuse, support the high and stable levels of antibody expression of the cells that produce the selected antibody, and are sensitive to a medium such as the HAT medium. The most preferred immortalized cell lines are murine myeloma lines, which can be obtained for example, from the Salk Institue Cell Distribution Center , San Diego, California and the American Type Culture Collection. American), Rockville, Maryland. The human myeloma and murine-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol ., 133 : 3001 (1984); Brodeur et al., " Monoclonal Antibody Production Techniques and Applications ", Marcel Dekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can be analyzed to see the presence of monoclonal antibodies directed against the PRO polypeptide of interest. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding analysis, such as radioimmunoassay (RIA) or enzyme linked immunosorbent analysis (ELISA). Such techniques are analyzes known in the art. The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem ., 107 : 220 (1980).

After identifying the hybridoma cells, the clones can be subcloned by limiting dilution procedures and can be grown by usual procedures [Goding, supra ]. Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle Medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo as ascites in a mammal.

Monoclonal antibodies secreted by subclones can be isolated or purified from means of culture or ascites fluid by procedures purification with conventional immunoglobulins, for example, Protein A-Sepharose, chromatography with hydroxyapatite, gel electrophoresis, dialysis or chromatography of affinity.

Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in US Pat. No. 4,816,567. The DNA encoding the monoclonal antibodies of the infection can be easily isolated and sequenced using conventional procedures (for example using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed in expression vectors, which are transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that otherwise do not produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the host cells. DNA can also be modified, for example, by replacing the coding sequence of the constant domains of human heavy and light chains instead of homologous murine sequences [US Pat. No. 4,816,567; Morrison et al., Supra ] or by covalent binding to the entire immunoglobulin coding sequence or part of the coding sequence of a non-immunoglobulin polypeptide. Such non-immunoglobulin polypeptide can be substituted by the constant domains of an antibody of the invention, or they can be substituted by the variable domains of a combination site with the antigen of an antibody of the invention to create a chimeric bivalent antibody.

The antibodies can be monovalent antibodies. Procedures for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of the immunoglobulin light chain and a modified heavy chain. The heavy chain is generally truncated at any point in the Fc region to avoid crosslinking of the heavy chain. Alternatively, the relevant cysteine residues are replaced with another amino acid residue or removed to avoid crosslinking .

In vitro procedures are also suitable for preparing monovalent antibodies. The digestion of antibodies to produce their fragments, particularly Fab fragments, can be achieved using standard techniques known in the art.

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C. Humanized antibodies

The anti-PRO polypeptide antibodies of the invention may additionally comprise humanized antibodies or human antibodies. Humanized forms of non-human antibodies (eg, murine) are chimeric immunoglobulins, immunoglobulin chains or their fragments (such as Fv, Fab, Fab ', F (ab) 2 or other sub-sequences of antigen-binding antibodies) containing minimal sequences derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins (receptor antibody) in which residues of a complementary determining region (CDR) of the receptor are replaced with residues of a CDR of non-human species (donor antibody) such as mouse, rat or rabbit having the specificity, affinity and capacity desired. In some examples, the Fv structure residues of the human immunoglobulin are replaced with the corresponding non-human residues. Humanized antibodies may further comprise residues that are neither found in the receptor antibody nor in the CDR or imported structural sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a sequence of a consensus human immunoglobulin. The optimally humanized antibody will comprise at least a portion of a constant region of an immunoglobulin (Fc), typically that of a human immunoglobulin [Jones et al., Nature , 321 : 522-525 (1986); Riechmann et al., Nature , 332 : 323-329 (1988); and Presta, Curr. Op. Struct. Biol ., 2 : 593-596 (1992)].

Procedures for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into that of a source that is not human. These non-human amino acid residues often refer to "import" residues, which are typically taken from a variable "import" domain. Humanization can be performed essentially following the procedure of Winter et al. [Jones et al., Nature , 321 : 522-525 (1986); Riechmann et al., Nature , 332 : 323-327 (1988); Verhoeyen et al., Science , 239 : 1534-1536 (1988)], substituting murine CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567), in which substantially less than one intact human variable domain has been replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly FR residues are replaced by residues of murine antibody analog sites.

Human antibodies can also be produced using several techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol ., 227 : 381 (1991); Marks et al., J. Mol. Biol ., 222 : 581 (1991). The techniques of Cole et al., And Boerner et al., Are also available for the preparation of human monoclonal antibodies (Cole et al., " Monoclonal Antibodies and Cancer Therapy ", Alan R. Liss , p. 77 (1985) and Boerner et al., J. Immunol ., 147 (1) : 86-95 (1991)].

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D. Bispecific Antibodies

Bispecific antibodies are antibodies monoclonal, preferably human or humanized, that have binding specificities for at least two different antigens. In the case at hand, one of the specificities of union is by the PRO polypeptide, the other is for any other antigen, and preferably by a cell surface protein or receptor or receiving subunit

Procedures for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the coexpression of two pairs of heavy-light / light-heavy chains of immunoglobulins, where the two heavy chains have different specificities [Milstein and Neck, Nature , 305 : 537-539 (1983 )]. Due to the random variety of heavy and light immunoglobulin chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is normally achieved by affinity chromatography steps. Similar procedures are described in WO93 / 08829, published May 13, 1993, and in Traunecker et al., EMBO J. , 10 : 3655-3659 (1991).

The variable domains of the antibody with the desired binding specificities (antibody-antigen combination sites) can be fused to sequences of the constant domain of the immunoglobulin. The fusion is preferably with a constant domain of the heavy chain of an immunoglobulin, which comprises at least part of the hinge regions CH2 and CH3. It is preferred that they have the first constant region (CH1) of the heavy chain containing the site necessary for the binding of the light chain present in at least one of the fusions. The DNAs encoding the immunoglobulin heavy chain fusions, if desired, the immunoglobulin heavy chain, are inserted into separate expression vectors, and co-transfected into a suitable host organism. For additional details to generate bispecific antibodies see, for example, Suresh et al., " Methods in Enzymology ", 121 : 210 (1986).

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E. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently bound antibodies. It has been proposed to such antibodies, for example, to target cells of the immune system for unwanted cells [US Pat. No. 4,676,980], and for the treatment of HIV infection [WO91 / 00360; WO92 / 200373; EP03089]. It is contemplated that antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a sidulfide exchange reaction or forming a thioether bond. Some examples of reagents suitable for this purpose include iminothiolate and methyl mercaptobutyrimidate and those described, for example, in US Pat. No. 4,676,980.

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Uses of anti-PRO polypeptide antibodies

The anti-PRO polypeptide antibodies of the invention have several utilities. For example, anti-PRO polypeptide antibodies can be used in diagnostic assays for a PRO polypeptide, for example to detect expression in cells, specific tissues, or serum. Several diagnostic assay techniques known in the art can be used, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays performed in heterogeneous or homogeneous phases [Zola, " Monoclonal Antibodies: A Manual of Techniques " (Monoclonal Antibodies) : Manual of techniques), CRC Press. Inc. (1987) pp 147-158]. Antibodies used in diagnostic assays can be labeled with a detectable residue. The detectable residue should be able to produce, directly or indirectly, a detectable signal. For example, the detectable residue may be a radioactive isotope, such as H 3, C 14, P 32, S 35 or I 125, a fluorescent or chemofluorescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase, or horseradish peroxidase. Any method known in the art can be employed to conjugate the antibody to the detectable residue, including the procedures described in Hunter et al., Nature , 144 : 945 (1962); David et al., Biochemistry , 13 : 1014 (1974); Pain et al., J. Immunol. Meth ., 40 : 219 (1981); and Nygren, J. Histochem and Cytochem ., 30 : 407 (1982).

PRO anti-polypeptide antibodies are also useful for affinity purification of a PRO polypeptide a from a culture of recombinant cells or sources natural In this process, the antibodies against the polypeptide PRO are immobilized on a suitable support, such as Sephadex resin or filter paper, using procedures well known in the art. The immobilized antibody is then contacted with a sample containing the PRO polypeptide to be purified, a Once purified, it is washed with a suitable solvent that will remove substantially all of the sample material except the polypeptide PRO, which is bound to the immobilized antibody. Finally the support is washed with another suitable solvent that will release the PRO polypeptide of the antibody.

The following examples are offered with illustrative purposes only, and are not intended to limit the scope of The present invention in any way.

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Examples

Commercially available reagents mentioned in the examples were used according to the instructions of the manufacturer unless otherwise indicated. The source of the cells identified in the following examples, and throughout the descriptive report, through acquisition registration numbers ATCC is the American Type Cultures Collection American type), Rockville Maryland.

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Example 1 Extracellular domain homology research for identify novel polypeptides and their cDNA encoders

Extracellular domain (SCD) sequences (including the secretion signal sequence, if any) of approximately 950 known secretion proteins from the Swiss-Prot public database were used to search for EST databases. EST databases included public databases (for example, Dayhoff, GenBank), and private databases (for example, LIFESEQ?, Incyte Pharmaceutical, Palo Alto, CA). The search was performed using the BLAST or BLAST2 software (Altschul. And Gish, Methods in Enzimology , 266 : 460-80 (1996); http: //blast.wustl/edu/blast/README.html) as a comparison of ECD protein sequences up to a translation of 6 reading patterns of EST sequences. Those comparisons that with a Blast had a result of 70 (or 90 in some cases) or greater and that did not encode known proteins were grouped and assembled in consensus DNA sequences with the "phrap" program (Phil Green, University of Washington, Seattle , WA; (http://bozeman.mbt.washington.edu/phrap.docs/phrap.html).

Using this homology investigation of extracellular domains, consensus DNA sequences gathered related to the other EST sequences identified. In addition, the consensus DNA sequences obtained often (but not always) are extended using repeated cycles of BLAST and phrap to extend the consensus sequence as much as possible using the sources of EST sequences described above.

Based on the consensus sequences obtained as described above, oligonucleotides were then synthesized and used to identify by PCR a cDNA library containing the sequence of interest and to use as probes to isolate a clone from the full length coding sequence of a PRO polypeptide. Direct (.f) and reverse (.r) PCR primers are generally designed generally in the range of 20 to 30 nucleotides to produce a PCR product of approximately 100 to 1000 bp in length. Probe sequences (.p) are typically 40 to 55 bp in length. In some cases additional oligonucleotides were synthesized when the consensus sequence was greater than 1 to 1.5 kbp. To investigate several libraries in search of a full-length clone, the DNA of the libraries was investigated by PCR amplification, as in Ausbel et al., " Current Protocols in Molecular Biology ", with the pair of PCR primers. A positive library was then used
to isolate clones that encode the gene of interest using the oligonucleotide probe and one of the pair of primers.

The cDNA libraries that were used to isolate the cDNA clones were constructed by usual procedures using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with a dT oligo containing a NotI site, bluntly ligated with semi-phosphorylated SalI adapters, cut with NotI, and its size was determined approximately in a gel electrophoresis, and cloned in a certain direction into a vector of adequate cloning (such as pRKB or pRKD; pRK5B is a precursor to pRK5D that does not contain the SfiI site; see Holmes et al., Science , 253 : 1278-1280 (1991)) at the unique XhoI and NotI sites.

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Example 2 Isolation of cDNA clones encoding PRO245 human

A consensus DNA sequence was assembled in relation to the other EST sequences identified as described in Example 1 above, where the consensus sequence is denominated in the present invention as DNA30954.

Based on the consensus sequence DNA30954, it synthesized oligonucleotides to identify by PCR a cDNA library containing the sequence of interest and for use as probes to isolate a clone from the coding sequence Full length for PRO245.

A pair of PCR primers were synthesized (direct and inverse):

primer for direct PCR

5'-ATCGTTGTGAAGTTAGTGCCCC-3 ' (SEC ID No. 65)

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primer for reverse PCR

5'-ACCTGCGATATCCAACAGAATTG-3 ' (SEC ID No. 66)

Additionally, a probe of hybridization of synthetic oligonucleotides from the sequence consensus DNA30954 that had the following sequence of nucleotides:

Hybridization probe

5'-GGAAGAGGATACAGTCACTCTGGAAGTATTAGTGGCTCCAGCAGTTCC-3 ' (SEC. ID No. 67)

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In order to screen several libraries for one source of a full length clone, the DNA of the libraries through PCR amplification with the partner of PCR primers identified above. Then it used a positive library to isolate clones encoding the PRO245 gene using the oligonucleotide probe and one of the PCR primers.

The RNA for the construction of the libraries of CDNA was isolated from human fetal liver tissue. Sequencing of the DNA of the isolated clones as described above produced the full length DNA sequence for the PRO245 [referred to in the present invention as UNQ219 (DNA35638-1141)] and the derived protein sequence for the PRO245.

The complete nucleotide sequence of UNQ219 (DNA35638-1141) is shown in Figure 1 (SEQ ID NO.  63). Clone UNQ219 (DNA35638-1141) contains a only open reading frame with a start site of the apparent translation at nucleotide positions 89-91 and ending at the stop codon at nucleotide positions 1025-1027 (figure 1; SEC ID No. 63). The predicted polypeptide precursor has 312 amino acids in length (Figure 2). UNQ219 clone (DNA35638-1141) has been deposited with the ATCC on the 16th September 1997 and was assigned the ATCC deposit number 209265.

The amino acid sequence analysis of PRO245 full length polypeptide suggests that a part of the it has a 60% amino acid identity with the protein human c-myb and therefore can be a new member of the transmembrane protein receptor family tyrosine kinase

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Example 3 Use of the nucleic acid encoding the PRO polypeptide as hybridization probes

The following procedure describes the use of a nucleotide sequence encoding a PRO polypeptide as a hybridization probe.

The DNA comprising the coding sequence of a PRO polypeptide of interest as described herein Descriptive memory can be used as a probe or used as a base from which to prepare probes to look for homologous DNAs (such as those encoding natural variants of the PRO polypeptide) in human tissue cDNA libraries or genomic tissue libraries human.

Hybridization and washing of the filters that contain the DNA of libraries are made under the following highly restrictive conditions. Probe hybridization nucleic acid derivative encoding the PRO polypeptide radiolabelled with filters is done in a 50% formamide solution, 5X SSS, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, an aqueous solution of 0.1X SSC and 0.1% SDS at 42 ° C.

DNAs that have a sequence identity desired with the DNA encoding the sequence PRO polypeptide full length native can then be identified using usual techniques known in the art.

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Example 4 PRO Polypeptide Expression in E. coli

This example illustrates the preparation of a non-glycosylated form of a PRO polypeptide by recombinant expression in E. coli .

The DNA sequence encoding the desired PRO polypeptide is initially amplified using selected PCR primers. The primers must contain restriction enzyme sites that correspond to the restriction enzyme sites of the selected expression vector. A variety of expression vectors can be employed. An example of a suitable expression vector is pBR322 (derived from E. coli ; see Bolivar et al., Gene , 2 : 95 (1997)) which contains ampicillin and tetracycline resistance genes. The vector is digested with restriction enzymes and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences encoding an antibiotic resistance gene, a trp promoter, and a polyHis guide sequence (including the first six STII codons, polyHis sequence, and an enterokinase cut site), the region encoding the polypeptide Specific PRO, the lambda transcriptional terminator and an argU gene.

The ligation mixture is then used to transform a strain of E. coli selected using the procedures described in Sambrook et al., Supra . Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are selected. Plasmid DNA can be isolated and confirmed with restriction analysis and DNA sequencing.

Selected clones can be grown all the night in a liquid culture medium like LB supplemented with antibiotics The overnight cultivation can then be used to inoculate a larger scale crop. The cells are grown then until the desired optical density is reached, during which it is induced The expression promoter.

After culturing the cells for several hours more, the cells are collected by centrifugation. Sediment cell obtained in centrifugation can be solubilized using several agents known in the art, and the PRO polypeptide solubilized can then be purified using a column with a metal chelator in conditions that allow a strong union of the protein.

In WO99 / 14328, several polypeptides were expressed satisfactorily in E. coli in a form labeled with poly-His, using the following procedure. The DNA encoding the polypeptide was initially amplified using selected PCR primers. The primers contained sites for restriction enzymes that corresponded to the sites for restriction enzymes in the selected expression vector, and other useful sequences that provide reliable and efficient translation initiation, rapid purification on a column with a metal chelator, and proeolytic elimination with enterokinase. The PCR-amplified poly-His tagged sequences were then ligated into an expression vector, which was used to transform a host E. coli based on strain 52 (W3110 fuhA (tonA) lon galE rpoHts (htpRts) clpP (laclq )). The transformants were first developed in LB containing 50 mg / ml carbenicillin at 30 ° C with stirring until an OD 600 of 3-5 was reached. The cultures were then diluted 50-100 times in CRAP media (prepared by mixing 3.57 g of (NH 4) 2 SO 4, 0.71 g of sodium citrate x 2H 2 O , 1.07 g of KCl, 5.36 g of Difco yeast extract, 5.36 g of Sheffield SF Hycase (casein hydrolyzate) in 500 ml of water, as well as 110 mM MPOS, pH 7.3, 0.55% (w / v) glucose and 7 mM MgSO 4) and developed for approximately 20-30 hours at 30 ° C with stirring. Samples were extracted to verify expression by SDS-PAGE analysis and the entire culture was centrifuged to pellet the cells. The cell pellets were frozen until purification and refolding.

The E. coli mass of 0.5 to 1 liter of fermentations (6-10 g of sediments) was resuspended in 10 volumes (w / v) in a 7 M guanidine buffer, 20 mM Tris, pH 8.0. Solid sodium sulphite and sodium tetrathionate were added to make final concentrations of 0.1 M and 0.02 M, respectively, and the solution was stirred overnight at 4 ° C. This step results in the denaturation of the protein with all the cysteine residues blocked by sulfitolization. The solution was centrifuged at 40,000 rpm in a Beckman ultracentrifuge for 30 minutes. The supernatant was diluted with 3-5 volumes of metal chelator column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micrometer filters to purify. Depending on, the purified extract was loaded on a column with 5 mL Qiagen metal chelator equilibrated with column buffer with metal chelator. The column was washed with an additional buffer containing 50 mM imidazole (Calbiochem, Utrol Grade), pH 7.4. The protein was eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein were combined and stored at 4 ° C. Protein concentration was estimated by its absorbance at 280 nm using the extinction coefficient calculated based on its amino acid sequence.

Proteins were refolded by diluting the sample slowly in a freshly prepared refolding buffer compound by 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refolding volumes are they chose so that the final protein concentration was 50 at 100 micrograms / ml. The refolding solution was stirred gently at 4 ° C for 12-36 hours. The reaction of refolding stopped by adding TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution was filtered through a 0.22 filter micrometers and acetonitrile was added to a final concentration of 2-10% The refolded protein was chromatographed on a Poros R1 / H reverse phase column using a 0.1% mobile phase of TFA with elution with a gradient of acetonitrile of 10 to 80%. Be analyzed aliquots of fractions with an absorbance at 280 nm in polyacrylamide gels with SDS and fractions that were combined They contained homogeneous folded protein. Generally the species properly retracted from most proteins elute at the lowest concentrations of acetonitrile since those species they are the most compact with their hydrophobic interior protected from the interaction with the reverse phase resin. The species aggregates are usually eluted at higher concentrations of acetonitrile In addition to solving the misfolded forms of proteins in the desired way, the reverse phase stage eliminates the endotoxin of the samples.

Fractions containing proteins desired folds desired folds were pooled and the acetonitrile using a gentle stream of nitrogen directed at the solution. Proteins were formulated in 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by filtration gel using G25 Superfine (Pharmacia) resins balanced in the formulation buffer and sterile filtered.

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Example 5 PRO Polypeptide Expression in Mammalian Cells

This example illustrates the preparation of a form glycosylated of a desired PRO polypeptide by expression recombinant in mammalian cells.

The vector, pRK5 (see EP307,247, published in March 1989), is used as the expression vector. Optionally, the DNA encoding the PRO polypeptide is ligated into the pRK5 with restriction enzymes selected to allow the introduction of the PRO polypeptide DNA using ligation procedures such as those described in Sambrook et al., Supra . The resulting vector is called pRK5-PRO polypeptide.

In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown until they converge in tissue culture plates such as DMEM supplemented with fetal calf serum and optionally, nutritional components and / or antibiotics. Approximately 10 µg of pRK5-PRO polypeptide DNA is mixed with about 1 µg of the DNA encoding the VA RNA gene [Thimmappaya et al., Cell , 31 : 543 (1982)] and dissolved in 500 µl of Tris -1 mM HCl, 0.1 mM EDTA, 0.227 CaCl2. To this mixture, 500 µL of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO 4 are added dropwise and a precipitate is allowed to form for 10 minutes at 25 ° C. The precipitate is resuspended and added to the 293 cells and allowed to settle for approximately four hours at 37 C. The culture medium is aspirated and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for approximately 5 days.

Approximately 24 hours after transfections, the culture medium is removed and replaced with culture medium (only) or culture medium with cysteine-S 35 200 µCi / ml and methionine-S 35 200 µCi / ml. After one 12 hour incubation, the conditioned medium is collected, concentrated in a centrifuge filter, and loaded in a gel with 15% of SDS. The processed gel can be dried and exposed to a film over a period of time to reveal the presence of polypeptide PRO. Cultures containing transfected cells can continue incubation (in serum free medium) and the medium will test in selected bioassays.

In an alternative technique, the PRO polypeptide can be introduced into 293 cells temporarily using the dextran sulfate method described in Somparyrarc et al., Proc. Nati Acad. Sci ., 12 : 7575 (1981). 293 cells are grown to a maximum optical density in a rotating vessel and 700 µg of pRK5-PRO polypeptide DNA is added. The cells are first concentrated in the rotating vessel by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium and reintroduced into the rotating container containing the tissue culture medium, 5 µg / ml bovine insulin and 0.1 µg / ml of bovine transferrin. After approximately four days, the conditioned medium can be concentrated and purified by any selected procedure, such as dialysis and / or chromatographic column.

In another embodiment, the PRO polypeptides are can express in CHO cells. PRK5-polypeptide PRO can be transfected into CHO cells using known reagents as CaPO_ {4} or DEAE-dextran. As described previously, cell cultures can be incubated, and replaced the medium with culture medium (only) or medium with an isotope radioactive as methionine-S. After determining the presence of the PRO polypeptide, the medium of culture with serum-free medium. Preferably the cultures are incubated. for about 6 days and then concentrate and purify by any selected procedure.

PRO polypeptide labeled with an epitope is it can also express in CHO host cells. The PRO polypeptide is You can subclone out of the pRK5 vector. The subclone insert is can undergo PCR to merge into the reading frame with a epitope tag selected, such as a tag from poly-His in a Baculovirus expression vector. The PRO polypeptide insert labeled with poly-His can be subcloned below in a vector directed by SV40 containing a selection marker, such as DHFR to select stable clones. Finally the cells CHO can be transfected (as described above) with the vector directed by SV40. Marking can be done, such as described above, to verify the expression. TO then the culture medium containing the PRO polypeptide expressed labeled with poly-His can be concentrate and purify by any procedure selected, by as per affinity chromatography with quilato-Ni 2+ PRO245 was expressed in a manner satisfactory in CHO cells by an expression procedure transient and a stable expression procedure.

Stable expression in CHO cells was performed. using the following procedure. Proteins were expressed as an IgG (immunoadhesin) construct, in which the sequences encoders of soluble forms (eg domains extracellular) of the respective proteins were fused to a sequence of a constant region IgG1 containing the region hinge, domains CH2 and CH2 and / or is a form labeled with polyHis.

After PCR amplification, the respective DNAs were subcloned into a CHO expression vector using usual techniques as described in Ausubel et al., " Current Protocols in Molecular Biology ", Unit 3.16, John Willey and children (1997). The CHO expression vectors are constructed so that they have 5 'and 3' compatible restriction sites of the DNA of interest to allow convenient transport of the cDNAs. The expression vector used in CHO cells is as described in Lucas et al., Nucl. Acids Res ., 24 : 9 (1774-1779 (1996)), and uses the SV40 early promoter / enhancer to direct the expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression allows selection for stable maintenance of the plasmid after transfection.

Twelve micrograms of the desired plasmid DNA were introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect * (Quiangen), Dospei *, or Fugene * (Boehringer Mannheim). Cells were grown as described in Lucas et al., Supra . Approximately 3 x 10-7 cells were frozen in a vial for further growth and production as described below.

Blisters containing plasmid DNA are thawed by placing them in a water bath and mixed with a vortex The contents were taken with a pipette and transferred to a centrifuge tube containing 10 ml of medium and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells were resuspended in 10 ml of selective medium (PS20 filtered with by a 0.2 µm filter with 5% dialyzed bovine fetal serum and filtered with a 0.2 µm filter). They alicuotó then the cells in a 100 ml rotating container containing 90 ml of selective medium. After 1-2 days, the cells they were transferred to a 250 ml rotating container with 150 ml of selective growth medium and incubated at 37 ° C. After another 2-3 days, 3 x 10 5 were seeded cells / ml in rotating containers of 250 ml, 500 ml and 2000 ml. He cell medium was changed to fresh medium by centrifugation and resuspension in the production medium. Although you can use a suitable CHO medium, a medium was actually used described in US Pat. No. 5,122,469, published on 16 June 1992. 3 l of the rotating vessel were seeded with 1.2 x 10 6 cells / ml. On day 0, the pH and the number of cells. On day 1, a sample of the rotating vessel was taken and it began to bubble with filtered air. On day 2, he took a sample of the rotating vessel, the temperature was changed to 33 ° C, and 30 ml of glucose 500 g / l and 0.6 ml of antifoam were added to the 10% (for example, 35% polydimethylsiloxane emulsion, emulsion Medical degree Dow Corning 365). Throughout the production, it adjusted the pH as necessary to keep it approximately at 7.2. After 10 days, or until the viability decreased by below 70%, the cell culture was collected by centrifugation and filtered through a 0.22 µm filter. Filtering or stored at 4 ° C or loaded on columns for purification.

For constructions labeled with polyHis, proteins were purified using a column Ni-NTA (Qiagen). Before purification, it is added the imidazole to the conditioned medium to a concentration of  5 mM. The conditioned media were pumped to a column 6 ml Ni-NTA equilibrated with 20 mM Hepes, pH 7.4, containing 0.3 M NaCl buffer and 5 mM imidazole at a rate of flow of 4-5 ml / min at 4 ° C. After loading, it washed the column with additional equilibration buffer and eluted the protein with equilibration buffer containing 10 mM Hepes, NaCl 0.14 M and 4% mannitol, pH 6.8, with a G25 Superfinde column of 25 ml (Pharmacia) and stored at -80 ° C.

The constructions with immunoadhesin (which contain Fc) were purified from the conditioned medium as described below. The conditioned medium was pumped on a 5 ml Protein A column (Pharmacia) that had equilibrated with 20 mM sodium phosphate buffer, pH 6.8. After loading, it washed the column abundantly with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is neutralized immediately by collecting 1 ml fractions in tubes that contain 275 µl of 1 M Tris buffer, pH 9. The salts of the solution with highly purified protein in buffer storage as described above for proteins polyHis. Homogeneity in polyacrylamide gels was assessed with SDS and by sequencing of the amino terminal end by Edman degradation.

PRO2945 was also expressed temporarily satisfactorily in COS cells.

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Example 6 PRO polypeptide expression in yeasts

The following procedure describes the expression recombinant of a desired polypeptide in yeasts.

First, expression vectors were constructed for intracellular production or secretion of PRO polypeptides with the ADH2 / GAPDH promoter. The DNA encoding a desired PRO polypeptide, a selected signal peptide and the promoter are introduced into the appropriate restriction enzyme sites in the selected plasmid to direct intracellular expression of the PRO polypeptide. For secretion, the DNA encoding the PRO polypeptide can be cloned into the selected plasmid, together with a DNA encoding the ADH2 / GAPDH promoter, the guide sequence /
alpha factor secretion signal, and adaptive sequences, if necessary, for the expression of the PRO polypeptide.

Yeast cells, such as the strain of AB110 yeast, can be transformed with plasmids from expression described above and can be grown in media selected fermentation. The supernatant of cells transformed by acid precipitation 10% trichloroacetic and by separation on a polyacrylamide gel with SDS (SDS-PAGE), followed by staining of the Coomassie blue dye gels.

Recombinant PRO polypeptide can be isolated then purify by removing yeast cells from fermentation medium by centrifugation and then concentrating the medium using cartridge filters. The concentrate that contains the PRO polypeptide can be further purified using resins of Selected column chromatography.

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Example 7 Expression of PRO polypeptides in infected insect cells with Baculovirus

The following procedure describes the expression PRO polypeptide recombinant in infected insect cells with Baculovirus.

The desired PRO polypeptide is fused in the 5 'direction of an epitope tag contained with a vector of Baculovirus expression. Such epitope tag includes tags polyHis and immunoglobulin tags (such as Fc or IgG regions). Be they can employ a variety of plasmids, including plasmids commercially available plasmid derivatives such as pVL1393 (Novagen). Briefly, the PRO polypeptide or the desired portion of the PRO polypeptide (as the sequence encoding the domain extracellular of a transmembrane protein) is amplified by PCR with primers complementary to the 5 'and 3' regions. The 5 'gazebo may incorporate restriction enzyme sites (selected) flanking. The product is then digested with those enzymes of restriction and is subcloned into the expression vector.

The recombinant baculovirus is generated by co-infecting the above plasmid and BaculoGold ™ virus (Pharmigen) DNA in Spodoptera frugiperda ("Sf9") (ATCC CRL 1711) cells using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28 ° C, the released viruses are collected and used for further amplifications. Viral infection and protein expression is performed as described in O'Reilley et al., "Baculovirus expression vectors: A laboratory Manual", Oxford, Oxford University Press (1994) .

The expressed polyHis-labeled PRO polypeptide can then be purified, for example with affinity chromatography with Ni 2+ chelate as described below. Extracts will be prepared from Sf9 cells infected with the recombinant virus as described in Rupert et al., Nature , 362 : 175-179 (1993). Briefly, the Sf9 cells are washed, resuspended in a sonication buffer (25 ml of Hepes, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicate twice for 20 seconds on ice. The sonicates are clarified by centrifugation, and the supernatant is diluted 50 times in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45? Filter. mum in diameter An agarose column is prepared with Ni2 + -NTA (commercially available from Qiagen) in a 5 ml support volume, washed with 25 ml of water and equilibrated with 25 ml of loading buffer. The filtered cell extract is loaded into the column at 0.5 ml per minute. The column is washed to an A280 reference base with loading buffer, at which point fraction collection begins. The column is then washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl; 10% glycerol, pH 6.0), which elutes the non-specifically bound proteins. After reaching the reference base at A 280 again, the column is developed with an imidazole gradient from 9 to 500 mM in the secondary wash buffer. Fractions of one ml are collected and analyzed by a polyacrylamide gel with SDS (SDS-PAGE) and silver staining or Western blot with Ni2 + -NTA conjugated to alkaline phosphatase (Qiagen). Fractions containing the His 10-labeled PRO polypeptide are combined and dialyzed against loading buffer.

Alternatively, purification of PRO polypeptide labeled with IgG (or labeled with Fc) can be perform using known chromatography techniques, including, by example, column chromatography of protein A or protein G.

PRO245 was expressed satisfactorily in Sf9 or high5 insect cells infected with baculovirus. While that the expression was actually performed on a scale of 0.5-2 L, you can easily increase the scale for larger preparations (for example, 8 L). The proteins as a construction of IgG (immunoadhesin), in which the extracellular region of the protein was fused with a sequence of constant region of IgG that contained the hinge region, the domains CH2 and CH3 and / or forms labeled with poly-His.

After PCR amplification, the respective coding sequences were subcloned into a Baculovirus expression vector (pb.PH.IgG for fusions with IgG and pb.PH.His for polyHis-tagged proteins), and the vector and the co-transfected BaculoGold® (Pharmigen) baculovirus DNA in Spodoptera frugiperda 105 ("Sf9") cells (ATCC CRL 1711), using Lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmigen), with modified polyiligator regions to include His or Fc tag sequences. The cells were grown in Hink TNM-FH medium supplemented with 10% FBS (Bovine Fetal Serum) (Hyclone). The cells were incubated for 5 days at 28 ° C. The supernatant was collected and subsequently used for the first viral amplification by infecting Sf9 cells in Hink TNM-FH medium supplemented with 10% FBS (Bovine Fetal Serum) at an approximate multiplicity of infection (MDI or MOI). of 10. The cells were incubated for 3 days at 28 ° C. The supernatant was collected and the expression of the constructs in the baculovirus expression vector was determined by batch binding of 1 ml of supernatant to 25 ml of Ni-NTA beads (QIAGEN) for histidine-tagged proteins or Protein A beads -Sepharose CL-4B (Pharmacia) for IgG-tagged proteins, followed by polyacrylamide gel analysis (SDS-PAGE) compared to a known concentration of protein standard by Coomassie blue staining.

The first supernatant was used viral amplification to infect a rotating culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) to an MDI of approximately 0.1. The were incubated cells for 3 days at 28 ° C. It was collected and filtered on supernatant Batch binding and gel analysis were repeated. polyacrylamide with SDS (SDS-PAGE), as outside necessary, until the expression of the culture was confirmed rotary.

The conditioned medium was collected for Transfected cells (0.5 to 3 L) by centrifugation to remove the cells and filtered through 0.22 micrometer filters. For constructions labeled with polyHis, the protein construction with a Ni-NTA column (Qiagen). Before purification, imidazole was added to the medium conditioned at a concentration of 5 mM. Conditioned media were pumped into a 6 ml Ni-NTA column equilibrated with 20 mM Hepes, pH 7.4, buffer with 0.3 M NaCl e 5 mM imidazole at a flow rate of 4-5 ml / m at 4 ° C. After loading, the column was washed with buffer additional equilibrium and protein eluted with buffer equilibrated with 0.25 M imidazole. Salts were removed from the highly purified protein and stored in a buffer of equilibrated with 10 mM Hepes, NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfinde column (Pharmacia) and stored at -80 ° C.

The constructions with immunoadhesin (which contain Fc) were purified from the conditioned medium as described below. The conditioned medium was pumped in a 5 ml Protein A column (Pharmacia) that had been equilibrated with 20 mM sodium phosphate buffer, pH 6.8. After loading, the protein was abundantly neutralized with buffer of equilibrated before elution with 100 mM citric acid, pH 3.5. The eluted protein was neutralized immediately by collecting fractions 1 ml in tubes with 275 ml of 1M Tris buffer, pH 9. Subsequently the protein salts were removed highly purified and stored in storage buffer as described above for proteins labeled with polyHis. The protein homogeneity was verified by gel electrophoresis of polyacrylamide with SDS (EGP) and amino end sequencing terminal by Edman degradation.

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Example 8 Preparation of antibodies that bind to polypeptides PRO

This example illustrates the preparation of monoclonal antibodies that can specifically bind to a PRO polypeptide.

Techniques for producing monoclonal antibodies are known in the art and are described, for example, in Goding, supra . Immunogens that may be employed include the purified PRO polypeptide, fusion proteins containing the polypeptide and cells expressing the recombinant PRO polypeptide on the cell surface. Those skilled in the art can perform immunogen selection if excessive experience.

Mice, such as Balb / c, are immunized with the Immunogen PRO polypeptide emulsified in Freund's adjuvant complete and injected subcutaneously or intraperiotoneally in an amount of 1-100 micrograms. Alternatively,  the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal on the back foot pads. It is then stimulated to mice immunized 10 to 12 days later with an immunogen additional emulsified in the selected adjuvant. From then, for several weeks, you can also stimulate the mice with additional immunizing injections. Can be obtained serum samples from mice periodically by taking retroorbital blood to perform ELISA assays to detect PRO polypeptide antibodies.

After a quantity of adequate antibodies, animals can be injected "positive" for intravenous injection antibodies PRO polypeptide end. Three or four days later, it is sacrificed to mice and spleen cells are collected. The spleen cells are then fuse (using 35% polyethylene glycol) to a line murine myeloma cell as P3X63AgU.1, available at ATCC, No. CRL 1597. Fusions generate hybridoma cells that can proliferate in tissue culture plates with 96 wells with medium HAT (hypoxanthine, aminopterin and thymidine) to inhibit proliferation of non-fused cells, myeloma hybrids, and Spleen cell hybrids.

Hybridoma cells can be analyzed in an ELISA to determine reactivity against the PRO polypeptide. The determination of "positive" hybridoma cells that secrete the desired monoclonal antibodies against the polypeptide PRO depends on the experience in the field.

Positive hybridoma cells can be inject intraperitoneally into syngeneic Blab / c mice to produce ascites containing anti monoclonal antibodies PRO polypeptide. Alternatively, hybridoma cells will they can grow in flasks or tissue culture screw bottles. The purification of the monoclonal antibodies produced in the ascites can be accomplished using precipitation with sulfate ammonium, followed by gel exclusion chromatography. Alternatively, affinity chromatography can be used. based on the antibody binding to protein A or G.

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Example 9 PRO chimeric polypeptides

PRO polypeptides can be expressed as chimeric proteins with one or more polypeptide domains additional added to facilitate the purification of the protein. Such purification facilitation domains include, but are not limited to metal chelating peptides as modules of histidine-tryptophan that allow purification with immobilized metals, protein A domains that allow the purification with immobilized immunoglobulin, and the domain used in the extension / affinity purification system FLAGS ™ (Immunex Corp., Seattle Wash.). The inclusion of a binding sequence that can be cut as Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between the domain of purification and sequence of the PRO polypeptide may be useful for facilitate the expression of the DNA encoding the PRO polypeptide.

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Example 10 Purification of PRO polypeptides using antibodies specific

Recombinant PRO polypeptides can be purify by a variety of usual techniques in the Protein purification technique. For example, the PRO propolypeptide, mature PRO polypeptide, or PRO prepolypeptide by affinity chromatography using antibodies specific for the PRO polypeptide of interest. In general, a column of immunoaffinity is constructed by covalently binding the antibody PRO anti polypeptide to an activated chromatographic resin.

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Polyclonal immunoglobulins are prepared to from serum precipitation with ammonium sulfate or by Purification in immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, the antibodies Monoclonal preparations are prepared from mouse ascites fluid by precipitation with ammonium sulfate or chromatography on Protein A immobilized. A partially purified protein is covalently bound to the chromatographic resin as SEPHAROSE? CnBr-activated (Pharmacia LKB Biotechnology) The antibody binds to the resin, the resin and the derived resin is washed according to the instructions of the maker.

Said immunoaffinity column is used in the PRO polypeptide purification by preparing a fraction of cells containing the PRO polypeptide in soluble form. This preparation is obtained by solubilizing all cells or a fraction subcellular obtained via differential centrifugation by means of adding a detergent or by other procedures very known in the art. Alternatively, the PRO polypeptide soluble containing a signal sequence can be secreted in a useful amount to the medium in which the cells grow.

A preparation containing a PRO polypeptide soluble is passed through the immunoaffinity column, and washed the column under conditions that allow preferential absorption of PRO polypeptide (eg, high ionic concentration buffers in the presence of detergent). Then, the column is eluted in conditions that alter PRO antibody / polypeptide binding (by example, a low pH buffer of approximately 2-3, or a higher concentration of an agent chaotropic such as urea or thiocyanate ion), and the polypeptide is collected PRO.

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Example 11 Drug selection

This invention is particularly useful for select compounds using PRO polypeptides or their fragments of union in any variety of drug selection techniques. He PRO polypeptide or fragment used in such a test may be free in solution, without joining a solid support, suspended in a cell surface, or located intracellularly. A Drug selection procedure uses host cells eukaryotes or prokaryotes that have been stably transformed with recombinant nucleic acids expressing the polypeptide or a PRO fragment. The drugs are selected against said cells transformed into competitive binding assays. Such cells, both in soluble and fixed form, can be used for tests of usual union. For example, the formation of complexes between the polypeptide or a PRO fragment and the agent that It is being tested. Alternatively, you can examine the decrease in the formation of complexes between the PRO polypeptide and its cell target or target receptors caused by the agent being testing.

Thus, the present invention provides procedures to select drugs or any other agent that may affect diseases or disorders associated with PRO polypeptide. These procedures comprise the contact of such agent with a PRO polypeptide or one of its fragments and the assay of (I) the presence of a complex between the agent and the polypeptide or a PRO fragment, or (II) the presence of a complex between the polypeptide or a PRO fragment and the cell, by very procedures known in the art. In such competitive union trials, the Polypeptide or PRO fragment is typically labeled. After from a suitable incubation the bound form of the polypeptide is separated or the PRO fragment, and the amount of free or unbound label attached to complex is a measure of the ability of the particular agent to bind to the PRO polypeptide or to interfere with the complex PRO / cell polypeptide.

Another technique for drug selection provides high performance of compound selection that they have the proper binding affinity to the PRO polypeptide and they described in detail in document WO84 / 03564, published on 13 September 1984. Briefly described, a large number is synthesized of different small peptide test compounds on a solid substrate, such as plastic needles or other surface. How I know has requested for a PRO polypeptide, the test compounds peptides are reacted with PRO polypeptides and washed. He PRO bound polypeptide is detected by methods well known in The technique. The purified PRO polypeptide can be coated directly on plates for use in selection techniques of drugs mentioned above. In addition, the antibodies do not neutralizers can be used to capture the peptide e immobilize it on the solid support.

This invention contemplates the use of tests of selection of competitive drugs in which antibodies neutralizers capable of binding to the PRO polypeptide specifically compete with a test compound at PRO polypeptide binding or its fragments. In this way, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with the PRO polypeptide.

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Example 12 Rational Drug Design

The objective of the rational drug design is to produce structural analogs of biologically active polypeptides of interest (i.e., a PRO polypeptide) or of small molecules with which they interact, for example, agonists, antagonists or inhibitors. Any of these examples can be used to model drugs that are more active or stable forms of the PRO polypeptide or that enhance or interfere with the function of the PRO polypeptide in vivo (cf. Hodgson, Bio / Technology , 9 : 19-21 (1991) ).

In one approach, the three-dimensional structure of the PRO polypeptide or of an inhibitor-PRO polypeptide complex is determined by X-ray crystallography, by computer modeling, or more typically, by a combination of the two approaches. The form and loads of the PRO polypeptide must be ascertained to elucidate the structure of the molecule and determine its active site (s). Less frequently, useful information related to the structure of the polypeptide can be achieved by modeling based on the structure of homologous proteins. In both cases, the relevant structure information is used to design molecules analogous to the PRO polypeptide or to identify effective inhibitors. Some useful examples of rational drug design may include molecules that enhance activity or stability as shown in Braxton and Wells, Biochemistry , 31 : 7796-7801 (1992) or that act as inhibitors, agonists or antagonists of native peptides as sample in Athauda et al., J. Biochem ., 113 : 742-746 (1993).

It is also possible to isolate an antibody with a specific target, selected by functional assay, as described above, and then resolve its crystalline structure. This approach, in principle, produces a drug core from which the design of a drug can be based. It is possible to encompass protein crystallography by generating anti-idiotype ( anti ids ) antibodies against a pharmacologically active, functional antibody. As a mirror image of a mirror image, one could expect that the anti ids binding site was analogous to the original receptor. The anti id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would act as a pharmacological core.

Under the present invention, there may be sufficient amounts of the PRO polypeptide available for Perform such analytical studies as X-ray crystallography. In addition, knowledge of the amino acid sequence of the PRO polypeptide provided herein provide guidance to those who use modeling techniques computer instead of or together with ray crystallography X.

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Example 13 Ability of the PRO245 polypeptide to inhibit proliferation of endothelial cell growth stimulated by the factor of vascular endothelial growth (VEGF)

The capacity of the PRO245 polypeptide was analyzed to inhibit stimulated endothelial cell proliferation by VEGF. Specifically, cells were plated endothelial adrenal cortical capillaries (ACE) bovine (del primary culture, 12-14 passes maximum) in plates of 96-well microtiters (Amersham Life Science) to a density of 500 cells / well per 100 µL in low DMEM in glucose, 10% calf serum, 2 mM glutamine, 1x pen / strept and fungizona, supplemented with 3 ng / ml of VEGF. Controls were put on plates in the same way, but VEGF was not included. Added one sample to be analyzed for the PRO polypeptide of interest in a volume of 100 µL for a final volume of 200 µL. The cells are incubated for 6-7 days at 37 ° C. The medium is aspirated and washed 1x cells with PBS. A mixture of acid phosphatase reaction (100 µL, 0.1 M sodium acetate, pH 5.5, 0.1% Triton-100, phosphate 10 mM p-nitrophenyl). After incubation for 2 hours at 37 ° C, the reaction was stopped by the addition of 10 µL of 1N NaOH. OD was measured on a plate reader microtiters at 405 nm. The controls were without cells, cells alone, cells + FGF (5 ng / ml), cells + VEGF (3 ng / ml), cells + VEGF (3 ng / ml) + TGF-? (1 ng / ml) and cells + VEGF (3 ng / ml) + LIF (5 ng / ml). (It is known that the TGF-? At a concentration of 1 ng / ml blocks 70-905 of cell proliferation stimulated by VEGF).

The results were evaluated by calculating the percentage of inhibition of cell proliferation stimulated with VEGF (3 ng / ml), determined by measuring acid phosphatase activity at OD 405 nm, (1) in relation to cells without stimulation and (2) in relation to the inhibition by TGF-? of reference of the activity stimulated by VEGF. The results, such as shown in table 2 below, they are indicative of the utility of the PRO145 polypeptide in cancer therapy and, specifically, in the inhibition of tumor angiogenesis. The numerical values (relative inhibition) shown in table 2 are determined by calculating the percentage of inhibition of VEGF stimulated proliferation by the PRO245 polypeptide in relation to cells without stimulation and then dividing that percentage by the percentage of inhibition obtained by TGF-? at 1 ng / ml which is known to block the 70-90% of cell proliferation stimulated by VEGF

TABLE 2

one

Example 14 In situ hybridization

In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences in cell or tissue preparations. It can be useful, for example, to identify gene expression sites, analyze the tissue distribution of transcription, identify and locate a viral infection, follow changes in the synthesis of a specific RNA and as an aid in chromosomal mapping.

Hybridization was performed in situ following an optimized version of the Lu and Gillett protocol, Cell Vision 1 : 169-176 (1994), using P 33-labeled ribosolves generated by PCR. Briefly, human tissues fixed with formalin and embedded in paraffin were cut, dewaxed, deprotected in proteinase K (20 g / ml) for 15 minutes at 37 ° C, and subsequently processed for in situ hybridization, as described in Lu and Gillett, supra . A non-coding UTP-labeled ribosonde [P 33] was generated from a PCR product and hybridized at 55 ° C overnight. The slides were bathed in an NTB2 nuclear tracking emulsion and exposed for 4 weeks.

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Synthesis of ribosondas with P 33

They were dried in a 6.0 µL vacuum centrifuge (125 mCi) of UTP-P 33 (Amersham BF 1002, SA <2000 Ci / mmol). To each tube with UTP-P33, The following ingredients were added:

2.0 µL of 5X transcription buffer

1.0 µL DTT (100 mM)

2.0 µl of NTP mixture (2.5 mM: 10 µl of each, GTP, CTP and 10 mM ATP + 10 µL of H2O)

1.0 µL of UTP (50 µM)

1.0 µl RNAse

1.0 µL of DNA template (1 µg)

1.0 µL of H2O

1.0 µL of RNA polymerase (for products of PCR T3 = AS, T7 = S, normally)

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The tubes were incubated at 37 ° C for one hour. 1.0 µL of DNase RQ1 was added, followed by incubation at 37 ° C during 15 minutes. 90 µL of TE (10 mM Tris pH 7.6; 1 mM EDTA pH 8.0), and the mixture was transferred with a pipette to a DE81 paper. The remaining solution was loaded in a unit of Microcon-50 ultracentrifugation, and centrifuged using program 10 (6 minutes). The filtration unit is Invested in a second tube and centrifuged using program 2 (3 minutes) After the final recovery pulse, 100 were added mul of TE, was transferred with a pipette 1 mul of product final to a DE81 paper and a count in 6 ml of Biofluor was made II.

The probe was run on a TBE / urea gel. Be added 1-3 µl of the probe or 5 µl of RNA Mrk III at 3 µl of loading buffer. After heating at 95 ° C heat the block for three minutes, the gel was placed immediately on ice. The wells were leveled, the sample, and ran at 180-250 volts for 45 minutes Saran coated gel was wrapped and exposed to an XAR film with a freezer intensification screen to -70ºC for one hour overnight.

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Hybridization-P 33 A. Pretreatment of frozen cuts

The slides were taken out of the freezer, placed in aluminum trays and thawed at temperature ambient for 5 minutes. The trays were placed in a incubator at 55 ° C for five minutes to reduce the condensation. The slides were fixed for 10 minutes at 4% of paraformaldehyde on ice in the fume hood, and washed in 0.5X SSS for 5 minutes, at room temperature (25 ml of SSC 20X + 975 ml of H 2 O SQ). After removing the 0.5 µg / ml proteinase K proteins for 10 minutes at 37 ° C (12.5 µL of 10 mg / ml stock solution in 250 ml buffer RNase without preheated RNase), the cuts were washed with SSC 0.5X for 10 minutes at room temperature. The cuts are dehydrated in 70% ethanol, 95%, 100%, 2 minutes in each one.

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B. Pretreatment of paraffin cuts

The paraffin was removed from the slides, placed in H 2 O SQ, and rinsed twice in 2X SSC to room temperature, for 5 minutes each time. The removed Cut proteins with 20 µg / ml proteinase K (500 µl of 10 mg / ml in 250 ml of RNase buffer without RNase, 37 ° C, 15 minutes) for human embryo, or K 8X proteinase (100 µl in 250 ml RNase buffer, 37 ° C, 30 minutes) for tissues with formalin. Subsequent rinsing in 0.5X SSC and dehydration were performed as described above.

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C. Prehybridization

The slides were prepared in a box of plastic coated with Box buffer (SSC 4X, 50% formamide) for saturated filter paper. The tissue was covered with 50 µl of hybridization buffer (3.75 g dextran sulfate + 6 ml of H 2 O SQ), stirred with a vortex and heated in the microwave for 2 minutes with the lid loosened. After cooling in ice, 18.75 ml of formamide, 3.75 ml of SSS 20X and 9 ml were added of H2O SQ, the tissue was agitated in the vortex well and incubated at 42 ° C for 1-4 hours.

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D. Hybridization

1.0 x 10 6 cpm probe and 1.0 were heated tRNA (50 mg / ml stock solution) per slide at 95 ° C for 3 minutes. The slides were cooled on ice, and 48 were added µl of slide hybridization buffer. After stirring with the vortex, 50 µl of P 33 mixture was added to 50 µl of prehybridization on the slide. The slides are incubated overnight at 55 ° C.

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E. Washes

2 10-minute washes were performed with SSS 2X, EDTA at room temperature (400 ml of SSC 20X + 16 ml of EDTA 0.25 M, Vf = 4 l), followed by a treatment with RNase A at 37 ° C for 30 minutes (500 µl of 10 mg / ml in 250 ml of buffer RNase = 20 µg / ml), the slides were washed twice during 10 minutes with SSC 2X, EDTA at room temperature. The conditions Restrictive washings were 2 hours at 55 ° C, 0.1X SSC, EDTA (20 ml of 20X SSC + 16 ml of EDTA, Vf = 4l).

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F. Oligonucleotides

In situ analysis was performed on the DNA sequence described in the present invention. The oligonucleotides used for this analysis were the following.

DNA35638-1141 (PRO245)

p1 5'-GGATTCTAATACGACTCACTATAGGGCGGGAAGATGGCGAGGAGGAG-3 ' (SEC ID No. 358) p2 5'-CTATGAAATTAACCCTCACTAAAGGGACCAAGGCCACAAACGGAAATC-3 ' (SEC ID NO. 359)

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G. Results

In situ analysis was performed on the DNA sequence described in the present invention. The results of this analysis are as follows.

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DNA35638-1141 (PRO245)

Expression was observed in the covering endothelium a subgroup of fetal and placental vessels. The expression Endothelial was confined to these tissue blocks. It was also observed expression in the cells of the intermediate trophoblast of the placenta. The rest of the tissues were negative.

Fetal tissues examined (weeks E12-E15) include : placenta, umbilical cord, liver, kidney, adrenal glands, thyroid, lungs, heart, large vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, marrow spinal, body wall, pelvis and lower limb.

Adult tissues examined : liver, kidney, adrenal glands, myocardium, aorta, spleen, lymph node, pancreas, lung, skin, cerebral cortex (rm), hippocampus (rm), cerebellum (rm), penis, eye, bladder, stomach, gastric carcinoma, colon, colonic carcinoma, thyroid (chimpanzee), parathyroid (chimpanzee), ovary (chimpanzee) and chondrosarcoma, liver damage induced by acetominophen and liver cirrhosis.

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Material deposit

The following materials have been deposited with the American Type Culture Collection American), 12301 Parklawn Drive, Rockville, MD, USA (ATCC):

2

These deposits were made under the provisions of the Budapest Treaty on recognition international deposit of microorganisms for the purpose of Patent procedure and regulations under it (Treaty of Budapest). This guarantees the maintenance of a crop Viable deposit for 30 years from the date of deposit. The ATCC will have deposits under the terms of the Treaty of Budapest, and will be subject to an agreement between Genentech, Inc. and the ATCC, which guarantees permanent availability and without restrictions on the offspring of the deposit culture to the public under the issuance of US Pat. relevant or under the setting public of any of the US patent applications or foreign, whichever comes first, and guarantees the availability of the offspring to one determined by the US It is authorized to Commissioner for patents and trademarks therein in accordance to 35 USC \ NAK 122 and the Commissioner's rules according to it (including 37 CFR \ NAK 1.14 with a particular reference to the 886 OG 638).

The assignee of this application has agreed that if a crop of the materials in deposit died or lost or destroyed when grown under conditions suitable, quickly replace the materials under notification with another equal. Material availability deposited is not built as a license to practice the invention in contravention of the rights granted by the authority or by any government under these laws on patents

The descriptive report previously written is considered sufficient to train any expert in the matter to practice the invention. The present invention is not limited in scope by the deposited construction, since the deposited embodiment is designed as a simple illustration of certain aspects of the invention and any of the constructions that are functionally equivalent fall within the scope of this invention. The deposit of material in this report descriptive does not constitute an admission that the description written in the present specification contained is inadequate to enable the practice of any aspect of the invention, including the best mode of it, nor is it built as limitation of the scope of the claims in the illustrations specific it represents. Moreover, several modifications of the invention in addition to those shown herein will be apparent to those skilled in the art from the above description and will fall within the scope of the claims added.

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References cited in the description

This list of references cited by the applicant is intended solely to help the reader and not It is part of the European patent document. Although he has put on maximum care in its realization, errors cannot be excluded or omissions and the EPO declines any responsibility in this respect.

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Claims (28)

1. Isolated nucleic acid encoding a PRO245 polypeptide, or complement thereof, in which the polypeptide: (i) comprises amino acids 1 to 312 of the Figure 2 (SEQ ID No. 64) or a mature form thereof; (ii) comprises a truncated or secreted form of natural form, a form of natural variant or an allelic variant natural polypeptide of Figure 2 (SEQ ID No. 64) and inhibits the proliferation of endothelial cell growth stimulated by VEGF, with the proviso that the polypeptide does not consist of amino acids 1-288 or 21-288 of the Figure 2 (SEQ ID No. 64); (iii) has at least 80% identity in the amino acid sequence with the amino acid sequence of the Figure 2 (SEQ ID No. 64) and inhibits growth proliferation of endothelial cells stimulated by VEGF, wherein said sequence identity is determined over the full length of said sequence, with the proviso that the polypeptide does not consist in amino acids 1-288 or 21-288 of Figure 2 (SEQ ID No. 64); or (iv) has at least 95% identity in the amino acid sequence with the amino acid sequence of the Figure 2 (SEQ ID No. 64) and inhibits growth proliferation of endothelial cells stimulated by VEGF, wherein said sequence identity is determined over the full length of said sequence. 2. Nucleic acid according to claim 1, part (iii), in which the level of identity is at least 85% 3. Nucleic acid according to claim 1, part (iii), in which the level of identity is at least 90% 4. Nucleic acid according to claim 1, in wherein said nucleotide sequence comprises a sequence of nucleotides consisting of the length coding sequence complete sequence shown in Figure 1 (SEQ ID No. 63), or the complement of it. 5. Nucleic acid according to claim 1, in wherein said nucleotide sequence comprises a sequence of nucleotides consisting of the sequence shown in figure 1 (SEQ ID No. 63), or the complement thereof. 6. Nucleic acid according to claim 1, which comprises the coding sequence for full length PRO245 of the DNA deposited under accession number ATCC 209265. 7. Vector comprising the nucleic acid according to any of the preceding claims. 8. Vector according to claim 7, attached operationally to control sequences recognized by a cell guest transformed with the vector. 9. Host cell comprising the vector according to claim 7 or claim 8. 10. Host cell according to claim 9, in which said cell is a CHO cell. 11. Host cell according to claim 9, wherein said cell is E. coli . 12. Host cell according to claim 9, in which said cell is a yeast cell. 13. Process to produce a PRO245 polypeptide comprising the culture of the host cell according to any of claims 9 to 12, under conditions suitable for the expression of said PRO245 polypeptide and recovery of said PRO245 polypeptide from cell culture. 14. PRO245 polypeptide isolated, wherein said polypeptide: (i) comprises amino acids 1 to 312 of the Figure 2 (SEQ ID No. 64) or a mature form thereof; (ii) comprises a truncated or secreted form of natural form, a form of natural variant or an allelic variant natural polypeptide of Figure 2 (SEQ ID No. 64) and inhibits the proliferation of endothelial cell growth stimulated by VEGF, with the proviso that the polypeptide does not consist of amino acids 1-288 or 21-288 of the Figure 2 (SEQ ID No. 64); (iii) has at least 80% identity in the amino acid sequence with the amino acid sequence of the Figure 2 (SEQ ID No. 64) and inhibits growth proliferation of endothelial cells stimulated by VEGF, wherein said sequence identity is determined over the full length of said sequence, with the proviso that the polypeptide does not consist in amino acids 1-288 or 21-288 of Figure 2 (SEQ ID No. 64); (iv) has at least 80% identity in the sequence with the amino acid sequence of encoded PRO245 by the nucleotide deposited under accession number ATCC 209265 e inhibits proliferation of endothelial cell growth stimulated by VEGF, in which said sequence identity is determines over the full length of said sequence, with the condition that the polypeptide does not consist of amino acids 1-288 or 21-288 of Figure 2 (SEC ID No. 64); or (v) has at least 95% identity in the amino acid sequence with the amino acid sequence of the Figure 2 (SEQ ID No. 64) and inhibits growth proliferation of endothelial cells stimulated by VEGF, wherein said sequence identity is determined over the full length of said sequence. 15. Polypeptide according to claim 14, part (iii), in which the level of identity is at least 85% 16. Polypeptide according to claim 14, part (iii), in which the level of identity is at least 90% 17. Chimeric molecule comprising a polypeptide according to any of the claims 14-16, fused to an amino acid sequence heterologous 18. Chimeric molecule according to claim 17, wherein said heterologous amino acid sequence is a Sequence of an epitope tag. 19. Chimeric molecule according to claim 17, wherein said heterologous amino acid sequence is a Fc region of an immunoglobulin. 20. Antibody that specifically binds to PRO245 polypeptide according to claim 14, part (i) or part (ii), with the proviso that the antibody is not an antibody that binds to a polypeptide that consists of amino acids 1-288 or 21-288 of Figure 2 (SEC ID No. 64). 21. Antibody according to claim 20, in the that said antibody is a monoclonal antibody. 22. Antibody that specifically binds to PRO245 polypeptide according to claim 14, part (i) or part (ii), which is a humanized antibody, a human antibody, a bispecific antibody, or a heteroconjugated antibody or has polyepitopic specificity. 23. Anti-PRO245 antibody antagonist, which specifically binds to the PRO245 polypeptide according to claim 14, part (i) or part (ii), and inhibits the activity of said polypeptide to inhibit growth proliferation of endothelial cells stimulated by VEGF. 24. Polypeptide according to any of the claims 14-16, for use in a method of treatment. 25. Polypeptide according to claim 24, for its use in cancer therapy. 26. Polypeptide according to claim 25, for its use in the inhibition of tumor angiogenesis. 27. Use of the polypeptide according to any of the claims 14-16 in the manufacture of a medicine for cancer therapy. 28. Use according to claim 27, wherein the Medication is for the inhibition of tumor angiogenesis.